biomolecules: structure and functions
TRANSCRIPT
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Introduction. Biochemistry explains the chemical processes that happen within an organism in order to maintain the
homeostasis. It studies the biomolecules, which are chemical structures characterized by a specific
composition (C, H, O, N, P, Ca and S).
Biomolecules can be divided in:
Large macromolecules: proteins, carbohydrates, lipids and nucleic acids;
Small molecules: metabolites, peptides.
BIOMOLECULES. There are four families of biomolecules:
1. Carbohydrates;
2. Lipids;
3. Nucleic acids;
4. Proteins.
1. CARBOHYDRATES can be divided, depending on the molecular weight, in
Simple: composed by a few molecules, for example:
o Monosaccharide: glucose (6C+1O, exons); fructose (pentons);
o Disaccharide: sucrose.
Complex, Polysaccharide (glycogen).
The functions of carbohydrates are:
a. Storage energy (glycogen);
b. Produce energy;
c. Produce the structure of a cell.
2. LIPIDS (FAT) are composed by a long chain of carbon; they are non-polar and hydrophobic. For example
triacylglycerol and phospholipid; this last one contain a phosphate which has a negative charge the
increase the charge of a molecule.
The functions of lipids are:
a. Storage energy;
b. Create a protection for the cell;
c. Messenger molecule.
3. NUCLEIC ACIDS are the repletion of a specific unit, composed by a phosphate group, a sugar (ribose or
2-deoxyribos) and a nitrogenous base. There are two different type of nucleic acids (DNA and RNA) with
different 3D structures.
The functions of carbohydrates are:
a. Hold genetic code;
b. The synthesis of protein.
4. PROTEINS are complex structures composed by the repletion of specific units, called amino acid; the
sequence of amino acids form the polypeptide chain, which is the first structure.
The functions of proteins are:
a. Structural;
b. Transport;
c. Chemical signals;
d. Movements;
e. Immune response;
f. Catalysis.
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WATER. Water is strictly connected with life as, for example, is the main solvent of the cells; moreover, in the prebiotic
era, the ultraviolet rays of sun have favoured reactions between inorganic molecules present in the
atmosphere by forming organic compounds, like amino acids.
Two molecule of hydrogen are link with a molecule of oxygen with a covalent bound, which
is a strong interaction impossible to destroy in physiological conditions.
The oxygen represents the negative pole of the molecule, because it has an higher
electronegativity compared to hydrogen; this means that, even if electrons are put in
common in the covalent bound, the electrons statistically spends more time near the
oxygen than near the hydrogen.
Water molecules can interact with other water molecules through hydrogen bounds,
which is a weak interaction, that can be easily changed in physiological conditions.
For this reason, charge or polar biomolecules are able to interact with water and can be
solubilized in it; this process is called hydratation and consists in the interaction between
ionized carboxyl groups and H of water and between protonated amine groups and O of
water. This kind of molecules are called hydrophilic compound, while hydrophobic
molecules are apolar and are soluble in organic solvents.
Amphipathic molecule are characterized by the presence of both polar or charged region and apolar region.
For example phospholipids are amphipathic molecule; in a polar solvent they can organize in a way to reduce
the interaction of the apolar region with the solvent. They can organize in micelle or bilayer.
OSMOSIS. Osmosis is the movement of solvent from low concentrated solution to high concentrated solution through
a semipermeable membrane (like cellular membrane) in order to achieve an equilibrium.
It is a colligative properties, which means that it depends only on solute concentration.
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WATER SELF-IONIZATION.
Water has a double behaviour: in a reaction with two molecules of water, one is able to lose one proton ion (becoming 𝐻+ + 𝑂𝐻−), the second eater molecule is able to become 𝐻3𝑂+.
The ionization constant 𝐾𝐶 is defined as the measure of the strength of an acid in solution and is the equilibrium constant for chemical reaction known as dissociation.
𝐾𝐶 =[𝐻3𝑂+][𝑂𝐻−]
[𝐻2𝑂]2= 3,2 108
The water ionic product 𝐾𝑊 is so defined as
𝐾𝑊 = [𝐻2𝑂]2𝐾𝐶 = [𝐻3𝑂+][𝑂𝐻−] = 10−14 @25°𝐶
And
𝐾𝑊 = [𝐻+][𝑂𝐻−] = 10−14
In pure water [𝐻3𝑂+] = [𝑂𝐻−] , so 𝐾𝑊 = [𝐻+][𝑂𝐻−] = 𝑥2 → [𝐻+] = [𝑂𝐻−] = 10−7𝑀
pH. A solution is considered:
Acidic: [𝐻+] > 1.0 10−7 𝑀 , which have an high concentration of proton ions; Neutral: [𝐻+] = 1.0 10−7 𝑀; Basic: [𝐻+] < 1.0 10−7 𝑀 , which have an high concentration of proton ions.
pH is defined as 𝑝𝐻 = −𝑙𝑜𝑔[𝐻+].
Moreover, it is possible to define 𝑝𝑂𝐻 = −log [𝑂𝐻−].
As
𝑘𝑊 = [𝐻+][𝑂𝐻−] = 10−14
− log([𝐻+] ∗ [𝑂𝐻−]) = −𝑙𝑜𝑔10−14
− log[𝐻+] − log[𝑂𝐻−] = 14
So
𝑝𝐻 + 𝑝𝑂𝐻 = 14
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ACID AND BASE
Acid compounds are able to release the proton ions to form the corresponding conjugate base.
Base compounds are able to accept a proton to form the corresponding conjugate acid.
Acids are able to release a proton ion, while bases are able to produce a negatively charged ion.
Base + strong acid → weak acid + weak base
The strength of an acid or of a base is related to the possibility to release proton ion or to release anion ion: strong acid/base have a complete dissociation.
The constant of dissociation is specific for each compound. When it is high (𝑘𝑎 >> 1), it means that the acid or the base is a strong compound.
On the contrary, weak acid/base have an incomplete dissociation, or an equilibrium.
By reading the dissociation constant it is possible to understand if the acid/base is strong or weak.
BUFFER SOLUTION.
The buffer is a solution of acid/conjugate base or base/conjugate acid characterized by constant and fixed pH value. That means that a small amounts of 𝐻+ or 𝑂𝐻− don’t change pH because they are neutralized reacting with conjugate base or conjugate acid respectively.
At the equilibrium, the weak acid is partially dissociate in a proton and the conjugate base; anyway, the addition of protons do not decrease the pH as the base links to it. Therefor the pH remains the same.
The buffering capacity is the ability to stabilize pH and is maximus if 𝑝𝐻 = 𝑝𝐾, but the buffer solutions work within the buffer window,
where [𝐴−]
[𝐻𝐴]< 10.
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Example of a buffer solution: the human blood.
The human blood is quite neutral and has pH = 7.3 - 7.4 which is maintained constant by a bicarbonate buffer (𝐻𝐶𝑂3−
/𝐻2𝐶𝑂3):
carbonic acid (weak acid) is able to dissociated in proton ions and bicarbonate.
Venous blood has a low concentration of oxygen and an high concentration of carbonic acid, produced by cells as waste. 𝐶𝑂2 is released into venous blood because it is a gas but is toxic so it has to be neutralized: this happens by the action of carbonic anhydrase (enzyme presents in the erythrocyte) which catalyses the reaction between carbon
dioxide with water into carbonic acid.
Normally, carbonic acid would dissociate into bicarbonate ions and hydrogen ions as the high concentration of 𝐶𝑂2 shift the buffer equilibrium towards these compounds. The increase of the concentration of hydrogen ions would decrease the pH; just thanks to the presence of hydrogen ions produced by cells the equilibrium is stable and the pH doesn’t change.
In lungs, the concentration of oxygen is high: this parameters move the equilibrium to the formation of carbon dioxide and water.
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LIFE AND THERMODYNAMICS
Living beings are member of closed circle where the mass of system remains constant over the time (mass is transformed but not eliminated) as postulated by principle of mass conservation (Lavoiser). Living beings are open systems, able to assume nutrients from environment and to release energy and waste into environment. They live in a system characterized by a general steady state: state of dynamic equilibrium typical of cyclic systems.
There are three thermodynamics law and each of them is able to introduce one different state function; a state function is a function which depends only on the initial and the finale conditions.
First law of thermodynamics. Conservation of energy: energy can neither be created nor destroyed. The total amount of energy in the universe is always conserved, or constant. Energy could be change into different form, but it is not possible to create or eliminate energy.
The internal energy is defined as the difference of heat (provided from environment) and work (done by system) and it is called enthalpy.
∆𝑈 = 𝑞 − 𝑤
Considering biological processes, it is possible to some characteristic: the pressure is the atmospheric pressure, so 𝑤 = 𝑃 ∆𝑉 in every biological system; the volume is normally constant, so 𝑃 ∆𝑉=0;
so in a biological process, the internal energy is the heat, defined as enthalpy ∆𝐻:
∆𝑈 = 𝑞𝑃 = ∆𝐻
In a reaction there is a variation of enthalpy, which is define as ∆𝐻 = 𝐻𝑓𝑖𝑛𝑎𝑙𝑒 − 𝐻𝑖𝑛𝑖𝑧𝑖𝑎𝑙𝑒
If ∆𝐻 is a positive value it means that the energy of the final product is higher than the initial, so in order to produce the final product the system has to acquire energy; this is defined an endothermic reaction. On the contrary, if ∆𝐻 is a negative value it means that the energy of the final product is lower than the energy of the initial one. There is a release of energy.
Second law of thermodynamics. The entropy, or disorder, of the universe must increase with every process that occurs. The measure of disorder is a state function called entropy.
∆𝑆 = ∆𝑆𝑠𝑖𝑠𝑡𝑒𝑚𝑎 + ∆𝑆𝑎𝑚𝑏𝑖𝑒𝑛𝑡𝑒 > 0
Entropy is strictly connected with the enthalpy because
∆𝑆𝑠𝑖𝑠𝑡𝑒𝑚𝑎 =𝑞
𝑇=
∆𝐻
𝑇
In biological condition, the temperature is fixed at 37°C.
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Normally, in the universe, the entropy increase, so the spontaneity is obtained when entropy increase; so if one reaction is characterized by a positive variation of entropy, the reaction could be spontaneous.
Third law of thermodynamics. Determination of reaction’s spontaneity
The relationship between entropy and enthalpy is given by the Gibbs free energy:
∆𝐺 = ∆𝐻 − 𝑇∆𝑆
When the Gibbs free energy is negative, a reaction is spontaneous and it is called exergonic. When the Gibbs free energy is 0, a reaction is at the equilibrium state. When the Gibbs free energy is positive, a reaction is not spontaneous and it is called endergonic as it requires energy.
Given a general reaction :
the variation of free Gibbs energy depends on free Gibbs energy at standard conditions (25°C and pression is atmospheric), and on the condition of the experiment (temperature, concentration and a constant of equilibrium of the reaction).
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Proteins. Proteins are biomolecules present in all living systems.
1828: beginning of biochemistry: organic compounds (urea) could be derived from simple inorganic
chemicals (ammonium cyanate)
1839: (Mulder) discovery of macromolecules, constituted by C-H-O-N, called proteins from Latin word
primarius or Greek god Proteus
1873: discovery of amino acids (AAs), the smaller units of proteins
1902: recognition of peptide bond, formed by condensation of free Aas
1926: crystallization of first enzyme (urease) and Pauling’s description of peptide bond geometry
1953: (Watson, Crick, Franklin and Wilkins) structure of DNA double helix
1958: (Kendrew) three-dimensional structure of proteins (haemoglobin)
1993: Human Genome Project has started
2000: first incomplete human genome sequence
2003: complete human genome map (all human genes are encoded)
Protein are formed by amino acids; there are only 20 AAs, combined in different positions in order to create
thousands of different proteins thanks to the variation in size and complexity.
The molecular mass is strictly connected to the differences in polypeptides chains; polypeptides chain is a
sequence of different amino acids, linked by peptide bound.
AMINO ACIDS. There are only 20 amino acids and all AAs are characterized by the same few different elements: C, H, N, O
and S.
The generale structure of a amino acid is:
Every amino acids contains
a. an acid group (Carboxyl group) which in specific condition is able to release a proton and form a
negative charge;
b. an basic group (Amino group) which in specific condition is able to link proton ions in order to form
a positive charge;
c. an H atom;
d. a lateral R chain, which characterize each amino acid.
In physiological condition, at the pH of 7,3 there is a complete dissociation of both chemical group (𝐶𝑂𝑂𝐻 releases a proton and forms a negative charge, 𝐻2𝑁 links a proton and form a positive charge). In this condition, without considering the different chemical groups, the amino acid is a compound where the number of negative charges is equal to the number of positive charges: therefore it is in his Zwitterion state. The Zwitterion state is an experimental condition where there is a balance between positive and negative charge and the net charge is 0.
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The isoelectric point (pI) is the specific pH on which the chemical compound is characterized by a complete balance between the different charges, therefore the protein has a neutral charge.
AMINO ACIDS PROPERTIES.
Acid – base properties. All amino acids (and all the proteins) are characterized by the buffer power: the two characterizing group are
able to neutralized every possible change of pH.
Considering an amino acids in a physiological condition,
a. If the pH decreases (e.g., due to catabolism) the
concentration of proton ions increases; the amino acids
try to maintain their number constant by linking the
proton ions to the amino groups. In this way, all amino
acids will be characterized by a positive charge in the amino group. On the other hand, also the
carboxyl group start to link the proton ions.
Therefore, in a condition of low pH, the amino acid has a positive charge.
b. If the pH increases, the concentration of proton ions
decreases; the amino acids try to maintain their number
constant by releasing the proton ions from the amino
groups. In this situation the amino group loses the
proton ions and the positive charge.
Therefore, in a condition of low pH, the amino acid has a negative charge.
In this two extreme condition, amino acids are able to lose their zwitterion state and assume one specific
charge.
For this reason, the calculation of pH it is possible through the same formula for buffer solutions, the
Peptide bonds. Amino acids are able to interact ach other in order to form peptide chains.
They can form a strong bond, which is defined a covalent bond; as the final product is a peptide, it is called
peptide bonds.
The carboxyl group of the first amino acid is able to interact
with the amino group of the second amino acid through a
condensation reaction (which release of one water
molecule).
The linkage of two amino acids forms a dipeptide.
The peptide bond is a planar chemical bonding, so it is able to
obtain a plane where it is possible to recognise the backbone
of the amino acids: the starting point presents a N terminus
positive charged while the ending point a C terminus negative
charged. In physiological condition, the backbone of a
polypeptide chain is in the zwitterion state.
In the lateral group it is possible to have other amino or carboxyl groups, so protein are not always in their
zwitterion state because it is possible that not all lateral chains’ charge are balanced.
Actually, the bond between the carbon atom and the hydrogen atom is not a simple bond.
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Chemical bond are not fixed condition, but are due to the
movement of electrons; in the peptide bond, the linkage occurs
between a carbon atom close to an oxygen atom, which is able to
attract the electrons involved in the bond. As the oxygen atom
attracts such electrons, it will be characterized by a partial negative
charge.
On the contrary, the nitrogen atom can balance this attraction by releasing some electrons which are
normally free from the interaction.
The result is that it is possible to recognize a partial negative charge on the Oxygen, a partial positive charge
on the Nitrogen and the presence of a double bonding between the Carbon of the first AA and the Nitrogen
of the second AA. So the peptide bond cant be define as a single chemical bond, due to the presence of a
partial double bond.
Cis e Trans.
The two atom of the peptide bound define a plane:
the atoms linked to the C-α can lay in the same
direction of the plane (CIS) or in different direction
(TRANS).
Normally, in vivo, there is the trans conformation:
the groups are in opposite direction considering the
plane formed by the double chemical bound, in order to reduce the repulsion between the groups.
AMINO ACIDS CLASSES. Amino acids could be divided into different groups by considering the chemical feature of all lateral groups.
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Glycine. 𝑅 = 𝐻. Glycine is the simplest amino acid and the lateral group is another
hydrogen atom. Therefore, glycine is the smallest amino acid: the biological consecution of
this is that glycine can confers flexibility to the protein.
R = ALYPHATIC SIDE CHAINS. The aliphatic side chain is formed by an high number of C linked together and
has a neutral charge. They are non polar and therefore are hydrophobic; they can perform the hydrophobic
interaction (weak interaction), which is involved in the formation of 3D structure.
Alanine. 𝑅 = −𝐶𝐻3. Alanine is still a small amino acid and is able to confer flexibility to the
structure, but no comparable to the flexibility which glycine confers.
R= HYDROXYL-CONTAINING SIDE CHAINS (-OH). These AAs, characterized by a hydroxyl side
group, are polar due to the electronegativity of the Oxygen. This group in not involved in the calculation of
pI of the protein because it is not able to release the proton.
The polarity of this group is important as there are weak interaction which occurs only between polar groups.
The presence of Hydrogen confers the possibility to perform hydrogen bonding, which is involved in the 3D
conformation of the protein.
The 3D structure of the protein is the result of weak interaction between side chain of AAs.
These AAs are point of post-traductional modification, which are chemical modification that occurs after the
synthesis of the protein e.g. (phosphorylation); these chemical reaction are able to link different molecules
to the amino acids inside a protein (sugar, methyl). A lot of protein are able to express their biological
structure only after the post-traductional modification, on the other hand some post-traductional
modification can transform a physiological protein to a pathological one.
The polarity of the hydroxyl group prevent the formation of net charges; therefore it is not
involved in the modification of pH.
Serine. 𝑅 = −𝐶𝐻2𝑂𝐻. the polarity prevent the formation of net charges.
R= ACID RESIDUES IN SIDE CHAINS. Acid AAs are characterized by the presence of another carboxyl group in
the side chain. The carboxyl group is able to release a proton ion in order to obtain a positive charge, so it is
important in order to calculate the pI. In vivo they are negative charged amino acids and are
involved in electrostatic relation with positive charged amino acids.
Moreover, they can produce the positive ions of our body; these are important because some
enzymes are able to catharize a reaction only after they are link to a positive ion.
Aspartate.
Glutamate.
R= AMIDE RESIDUES IN SIDE CHAINS. Amide AAs are characterized by the amide group, which is formed by N
and O. The oxygen is able to attract electrons and perform a negative pole, while the
nitrogen has a low electronegativity and can lose an electron and obtain a positive pole:
it is a polar group, therefore they are involved in hydrogen bonding.
The amide group is quite stable and make the whole amino acid stable in physiological
condition. For this reason they are not involved in post traductional modifications.
Asparagine.
Glutamine.
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R=SULPHUR ATOM. This group consists in just 2 AAs.
Cysteine. This AA is characterized by a short side chain with s thiol group (SH); this is similar to the hydroxyl
group and is quite reactive: is able to perform oxidation with another thiol in order
to obtain the disulphide bridge. One O is reduced into one water molecule by taking
two H and the result is a direct chemical bonding between the two S.
In physiological environment, this quiet strong bound is stable, but it is possible to reduce the bridge in order
to obtain 2 thiol group; this reduction is possible by changing the environmental condition: T, pH, or specific
reagent, which are able to catalyse the reaction (the most common reducing reagent are the
Mercaptoethanol or dithiothreitol (DTT)).
Methionine. In the Methionine isn’t possible to recognize the thiol group as S in directly boud to 𝐶𝐻3; this
reduce the chemical reactivity of S, so it isn’t possible to perform the disulphide bridges.
Therefore, M can be defined as an hydrophobic AA.
The presence of S can be useful to link metal complexes, which should be absent in
physiological environment; anyway the possibility to bind this toxic ions, is a way to
prevent the contamination and the pollution of a cell.
R= BASIC RESIDUES IN SIDE CHAINS. Basic AAs are able to obtain net positive charge: they can interact with
acid AAs in order to obtain ionic interaction.
They have a quite long side chain (4 or more C), so they are characterized by a quite high steric volume; for
this reason they are absent in little or flexible protein.
Lysine. This AA is formed by a side chain of 4 C and a final amino group.
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Arginine. This AA is formed by a side chain of 3 C and guanidine group. This is useful
to delocalize the possible positive charge in all 3 N atoms. For this
reason the arginine is normally completely protonated because
the charge is able to move on different atoms: this resonance
stabilize the chemical structure.
They can perform an ionic interaction (weak interaction which
occur during the 3D structure of a protein).
R= CYCLIC RESIDUES IN SIDE CHAINS. The cycle in the side chain conferees a high
sterical hindrance and prevent the movement of every other AAs: they are able to confers rigidity to the
protein, oppositely to the glycine; this can fix a specific 3D conformation. The biological structure of a protein
is due to a balance between flexibility and rigidity.
Proline. This AA is the smallest cyclic amino acid and contains a cycle of 5 atoms; one of these is the nitrogen,
which always bind a H atom: the proline can perform hydrogen binding.
It can also define hydrophobic as there due to the presence of net charge and of a
pole.
Histidine. Histidine also has a cycle of 5 atoms, but the carbon chain increases the number, so it has a major
sterical hindrance. This cycle can be protonated and form a net positive charge, but this
doesn’t affect the stability thank to the resonance: the charge can be localized on both
N atoms. In physiological conditions, the histidine is normally unprotonated.
It can perform hydrogen bounding.
R= AROMATIC RESIDUES. Aromatic cycle is formed by 6 C and has 3 double chemical bounding, which are
really strong links. Due to the resonance phenomenon, there are three similar structure by changing the
position of the double bond. This is possible because all atoms inside the cycle are C and makes the aromatic
ring a chemically inert structure.
Moreover, there are some electrons that are not involved in the double chemical bounding and which are
free inside an atom ( π electrons). They are able to move all around the structure: they confers the possibility
to perform the π-π interaction (weak interaction). This interaction can be performed with other aromatic
structure or with other structure with partial free electrons.
The presence of aromatic AAs prevent movements in the protein structure.
Tryptophan. This AA has a double cycle so his sterical hindrance stops the movement of polypeptide chains:
it can control the formation of a specific 3D structure.
One of the two cycle as an H atom: it can perform the π-π interaction and the hydrogen
bound.
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Tyrosine. The tyrosine is quite similar to phenylamine, but the aromatic side chain is linked to
the hydroxyl group: it can perform pi-pi interaction and hydrogen bounding interaction.
Hydrogen bond: hydroxyl groups, amino groups, N in a cycle linked to a hydrogen.
Ionic interaction: carboxyl groups + amide groups.
Hydrophobic interaction: C atoms.
AMINO ACID NOMENCALTURE.
The C-α is the one present in all AA, so the one linked to NH, COOH and H. if the side
chain contains other C, they are named β, γ, δ, ε etc…
THE ISOELECTIC POINT.
pKs are the dissociation constant present inside the amino acid. They are fixed parameter related to all
chemical group able to ionize (lose or acquire protons) themselves: it exist only for carboxyl and amino
groups. All amino acid are characterized by 2 pKs, one charactering the N terminal and one the C terminal,
just basic or acid AAs have 3 pKs as they are characterized by a charged side chain. pK of acid groups are
theoretically from 0 to 7, while pH of amino groups are between 7 and 14. Usually, the dissociation constants
are expressed as pK because K very low number, 𝑝𝐾 = − log(𝐾).
pI OF NEUTRAL AAs.
Glycine, characterized by H on the side chain, has just 2 pKs, one referred to the amino group and one
referred to the carboxyl group.
Every AA is characterized by a buffering power, so glycine could be present in different dissociated formulas
considering the different pH:
a. pH < pK of carboxyl group. At low pH, glycine is characterized by one positive charge. This is due to
the high concentration of protons, which is contrasted by the amino acid through the ionization of
both groups: the carboxyl group doesn’t dissociated and the amino group acquires an H.
b. pK of carboxyl group < pH < pK of amino group. At a pH within the pKs of the two groups, the amino
acid has a neutral charge. In this condition, for the carboxyl group the concentration of proton ions
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is not really high, so it release the proton ions to form one negative charge, while is very high for the
amino group, so it accept the proton ions.
In this condition, two different pKs should be considered in the pI formula: 𝑝𝐼 =2+9
2.
c. pH > pK of amino group. At high pH, at pH>9 the concentration of proton ions is low for both chemical
group so they release the proton. the Glycine is characterized by a negative charge.
As amino acids can be considered a buffer solution, pH is calculated with the Henderson-Hasselbach formula:
the pH depends on the pH and on the concentration of acid and basic compound. The formula is applied on
both equilibrium:
a. considering the first equilibrium, the pH is equal to the pK of
carboxyl group plus log of the conjugated base (Glycine, the one
obtained after the release of a proton ion);
b. considering the second equilibrium, the pK is the pK of the basic group
and the conjugated base is Glycine - .
As the condition is fixed pH is equal to pI and the concentration of Glycine + is the same to the concentration
of Glycine -.
This leads to the final formula: 𝑝𝐼 =1
2(𝑝𝐾𝑎1 + 𝑝𝐾𝑎2) .
pI ACIDic AAs. In acidic AAs there are 3 pK, so it is necessary to choose the 2 pK at which AA is neutral. The
lower acid pK is always referred to the C terminal (carboxyl group present in every AA), the second one the
acid group of the R acid group.
e.g. aspartate has 2 carboxyl groups and 1 amino group.
a. pH<1,8. The proton ions concentration is too high for every group, so the AA acquires and doesn’t
release protons.
both carboxyl groups are protonated and have a neutral charge as their pK are higher than
the pH,
amino group is protonated and acquires a positive charge as his pK is higher than the pH.
As the charge is not null, this pKs aren’t useful in order to calculate the pI.
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b. 1,8<pH<3,6. The proton ions concentration is too high for the amino groups and the carboxyl group
of the lateral chain.
the carboxyl group of the side chain is unprotonated and acquires a negative charge as his
pK is lower than the pH,
the carboxyl group of terminal is protonated as his pK is higher than the pH,
the amino group is protonated and acquires a positive charge as his pK is higher than the pH.
As the charge is null, the pI is calculated using this 2 pKs.
c. 3,65<pH<9,69. The proton ions concentration is too high for the amino group.
the carboxyl group of the side chain is unprotonated and acquires a negative charge as his
pK is lower than the pH,
the carboxyl group of the terminal is unprotonated and acquires a negative charge as his pK
is lower than the pH,
the amino group is protonated and acquires a positive charge as his pK is higher than the pH.
As the charge is not null, this pKs aren’t useful in order to calculate the pI.
d. pH>9,6. The proton ions concentration is low for the every group.
the carboxyl group of the side chain is unprotonated and acquires a negative charge as his
pK is lower than the pH,
the carboxyl group of the terminal is unprotonated and acquires a negative charge as his pK
is lower than the pH,
the amino group is unprotonated has a null charge.
As the charge is not null, this pKs aren’t useful in order to calculate the pI.
The pKs used to calculate the pI are the two acid pKs.
pI of BASIC AA. In basic AAs there are 3 pK, so it is necessary to choose the 2 pKs at which AA is neutral. The
highest pK is always referred to the amide group of the side chain.
e.g. lysine has a one carboxyl group and 2 amine groups.
a. pH<2,18. The proton ions concentration is too high for every group, so the AA acquires and doesn’t
release protons.
both amino groups are protonated and acquire a positive charge as their pK are higher than
the pH,
carboxyl group is protonated and has a nulla charge as his pK is higher than the pH.
As the charge is not null, this pKs aren’t useful in order to calculate the pI.
b. 2,18<pH<8,95. The proton ions concentration is too high for the amino groups.
both amino groups are protonated and acquire a positive charge as their pK are higher than
the pH,
the carboxyl group is unprotonated and acquires a negative charge as his pK is lower than
the pH.
17
As the charge is not null, this pKs aren’t useful in order to calculate the pI.
c. 8,95<pH<10,53. The proton ions concentration is too high for the amino group of the side chain.
the amino group of the side chain is protonated and acquires a positive charge as his pK is
higher than the pH,
the amino group of the terminal is unprotonated as his pK is lower than the pH,
the carboxyl group is unprotonated and acquires a negative charge as his pK is lower than
the pH.
As the charge is null, the pI is calculated using this 2 pKs.
e. pH>10,53. The proton ions concentration is low for the every group.
both amino groups are unprotonated and acquire a positive charge as their pK are lower than
the pH,
the carboxyl group is unprotonated and acquires a negative charge as his pK is lower than
the pH.
As the charge is not null, this pKs aren’t useful in order to calculate the pI.
The pKs used to calculate the pI are the two acid pKs.
In static condition, the pH is precise and fixed, but in
physiological condition the situation is completely
different because AAs interact each other and not all acid
o basic AAs are free.
AMINO ACID DERIVATES.
Amino acids form different biomolecules:
proteins,
nutrients,
neurotransmitter: GABA, dopamine: increase heart frequency and blood pressure,
hormones: thyroxine can regulate the metabolism of food,
histamine.
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Proteins
Proteins are composed by amino acids, which organize in different structures. Primary, secondary and third
structure are present in every protein, while the quaternary structure is typical of just high molecular weight
proteins.
The primary structure can be shorter or longer depending on the specific protein and is formed by the peptide
bond. It is a stable structure.
The secondary structure is the way the sequence of AAs interacts itself in order to form non-complex 3D
conformations.
The tertiary structure is the way the residues of the backbone interact each other to form a 3D structure.
The quaternary structure is present only in protein characterized by 2+ polypeptide chains.
Primary structure.
Primary structure
The primary structure is the sequence of the AAs and defines the biological function of the protein. The AAs
interact each other only through the peptide bond.
Some proteins are characterized by
a. Invariant regions: regions whose modification would affect the biological function;
b. Variant regions: regions whose modification wouldn’t affect the biological function (protein
polymorphism).
There are two types of modifications:
a. Conservative transition: substitution of 1+ AAs with other AAs with the same chemical-biological
feature. For example the substitution of 2 basic AAs or 2 hydrophobic AAs. This doesn’t change the
protein characteristics because the further structure are the same.
b. Unconservative transition: substitution of 2 completely different AAs (polarity, dimension, charge).
This affect the further structure
19
The AA sequence is read from left to right: it starts with the N-
terminal (not involved in the peptide bond), which is positive
charged and ends with a C-terminal, negative charged.
The primary structure isn’t a planar as the peptide bond is a
partial double bond, therefore residues will be in trans
position.
Torsion angles are formed between the C-alpha and N and C-
alpha and C.
Given a primary structure, it is possible to calculate the most probable
torsional angles, through the Ramachandrean plot. This represent all the
possible torsion angles: those in the blue areas are the probable one,
those in the green area are the less likely ones; others are not possible,
considering the sterical hindrance.
Secondary structure
The secondary structure is the spatial arrangement of polypeptide chain without considering side groups
conformation; is given by the weak interactions which happen in the backbone of the protein.
Some proteins have a well-arranged secondary structure (α-helix , β-sheet or β-turn), while the most of them
have an irregular secondary structure (random coil); in this last case, the Ramachandrean plot supplies a good
prediction of the torsion angles.
α - helix
Alpha helix is the most common motif found in proteins; it is characterized by a
precise number of AAs per turn (3,6) and a precise distance between the first and
the last AAs of the pitch (5,4A).
The most common winding in proteins is clockwise.
The α – helix is due to the hydrogen bonding between the O of carbonyl group and
the H binding to the N of the other AA.
This secondary structure is polar because the first amino group and the last
carboxyl groups are not involved in the formation of H bonding, for this reason they
form a dipole moment. This favours interactions with other polar structure.
Fibrous proteins (α – cheratins, collagen and silk protein like fibroin) always have
alpha-helix structure because is able to confers flexibility and strength to this kind
of protein. In these protein there is an high concentration of Gly (flexibility) and
Pro (rigidity)
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Β – sheet
The β-sheet structure is formed by an huge number of β
-strands, which are an elongated conformation with 2
AAs chains; these are not stable because of the low
number of local stabilizing interactions (hydrogen
bonding between the O of the carboxyl group and the H
of the amino one). β – strands can have a parallel or anti-
parallel arrangements.
The β – sheet is formed by 2+ β-strands interacting each other and connected by loops. Both parallel and
anti-parallel β-sheet exist.
β - TURN.
This structure can change the direction of one polypeptide chain; it’s formed
by a very small number of AAs. It is formed by 4 AA, where the first AA
interacts with the fourth one through an hydrogen bonding. Often, the third
AA is Gly and the second one is Pro: Gly favours the movement of the
polypeptide chain, while Pro stabilize the shape, conferring rigidity to the final
structure.
Tertiary structure
The tertiary structure is a result of more than one weak interaction (hydrophobic interactions, van der walls
interactions, disulphide bridges, ionic interactions, hydrogen bonds) which occurs between side chains. The
combination of these interaction results in the final 3D conformation.
Types of weak interaction:
1. Hydrogen bonding. Hydrogen bonding occurs between two side chains:
a. One able to accept an electron (e.g. hydroxyl group: tyrosine,
threonine and serine)
b. One able to release an electrons (e.g. amino group: glutamine and
asparagine ).
Side chains atoms can perform hydrogen bond with water, trapped within
the interior of protein.
2. Disulphide bridges. Disulphide bridges are possible just between two cysteines as they are characterized
by thiol groups. They are broken through a reduction which occurs only in extreme conditions, like high
temperature, acid pH, presence of reductants.
21
3. Van der Waals interactions. Van der Waals interactions are connected to the polar AAs present in the
side chain; it occurs when there is at least one polar group, so it can happen:
a. Between different permanent dipoles (orientation effect);
b. between permanent and temporary dipoles (induction effect);
c. between induced or temporary dipoles (dispersion effect or London force).
There isn’t the presence of a net charge.
4. Ionic interactions (electrostatic, ionic). Charge-charge
interactions occurse between different charge side chain, for
example acidic (carboxyl group, negative charged) and basic AAs.
5. Hydrophobic interaction. The hydrophobic interaction is
performed by hydrophobic side chains (e.g. aliphatic R).
Protein structural classification
α – protein. Characterized by the presence of α – helix; e.g. fibrous proteins;
β – proteins. Characterized by the presence of β – sheet; e.g. globular proteins (antibody);
α/β – proteins. Characterized by the presence of both structure: majority of proteins;
Irregular proteins. Characterized by the presence of few and small motifs: typical of low molecular
weight proteins, where the low number of AAs prevent the formation of a precise and regular
secondary structure;
22
β – barrels. Typical of protein with an high number of AAs: 8 parallel or antiparallel
β – sheet domains are able to interact each other and close in a circle linked by
loops.
α/β – barrels. Stable arrangement of 8 parallel or antiparallel β – sheet and α - helix
linked by loop.
Protein folding
The protein folding is a three-dimensional arrangement of polypeptide chain to obtain a biologically
functional conformation. Is the result of all weak interactions occurred inside the backbone and between
side chains. The final stable conformation is defined as native state (physiological conformation of a protein,
important to obtain a biological function).
The final 3D conformation is a direct consequence of the primary structure.
Different proteins are able to perform the same biological function: for example the Cytochrome c (small
protein involved in the transport of electrons associated to the inner membrane of mitochondrion) can be
characterized by different AAs: different primary structure can produce the same 3D structure and so he
same biological function. This is possible because also different AAs can be quite similar, so the same
percentage of the same groups of AAs (polar, aliphatic, charged…) performs the same weak interactions,
interact with the environment in the same way and form the same 3D structure.
Moreover, proteins classified as a unique family can be found in different species as characterized by the
same general composition.
Technologies able to describe protein secondary structure
X-ray crystallography.
The X-ray crystallography is able to identify atoms and
molecular structure of a crystal, analysing the diffraction
typical of chemical compound; the range of the diffraction
depends on the type and the number of chemical bonds inside
the crystal and the presence of weak interactions (secondary
and tertiary structure).
The first step of this procedure is creating the crystal of the
protein, which has to be sufficiently large, pure and regular.
Then, a software is able to convert electron density into
secondary structure.
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This technique is not precise with large macromolecules because they don’t crystalized in a precise way, while
is widely use with recombinant proteins.
Nuclear magnetic resonance (NMR) spectroscopy
NRM is able to determine the electronic structure of molecules by the exploitation
of magnetic properties of atomic nuclei, through the phenomenon called nuclear
resonance. The resonance frequencies is the energy that a molecule release after
the acquisition of electromagnetic energy.
First of all, the proteins are placed into a solution with specific isotopies, which
bound to the it. Then, the molecule is irradiate with an electromagnetic field, the
recognized resonance frequencies are transport into a specific graph. The
distances between the dots represent the distances between atoms. Given the plot and the primary
structure, specific software extrapolate the kind of structure.
This technique is used with just low molecular weight proteins.
Quaternary structure
The quaternary organization occurs just between different 3D polypeptide chains and is the result of weak
interactions that occur between different polypeptide chains (disulphide bridges, hydrophobic interactions,
ionic interactions and hydrogen bonds).
Is normally characterized by a rotational (cyclic) symmetry.
An example is the quaternary structure of the haemoglobin which
is characterized by 4 polypeptide chains.
The final 3D conformation is a result of specific weak interactions between polypeptide chains, so every 3D
conformation is important in order to form the biological functional protein.
Proteins with a quaternary conformation are characterized by other stable conformation, different from the
native one (the functional one), all with quite the same energy (metastable conformation). This prevent their
biological activity.
Suddenly, the presence of one specific metabolite (allosteric substrate) is useful in order to change the
conformation from inactive to the active one. This is called allosteric control.
PROTEIN STABILITY
The equilibrium between the functional and the unfolded protein is due to the possibility to change the free
Gibbs energy, which depend on the temperature and the entropy.
In physiological condition, these parameters are stable (T=37°, pH=7,35). Therefore, inside a cell there is only
one native conformation, but if the energy increases (e.g. heating) it is possible to confer more energy to the
protein useful to change weak interactions and so the final conformation.
Actually, in vivo this conditions are quite infrequent, but the modifications of the 3D conformation are
possible thanks to the electrons movement: this is called conformational breathing.
In vitro, it is possible to use some agents to change the 3D conformation:
Detergent: they interact with apolar side chains preventing hydrophobic interaction and folding;
Denaturants: urea and guanidine hydrochloride disrupt hydrophobic interactions because they are
able to increase water solubility of polar side chains.
These changes are possible thanks to the allosteric substrates, able to change the type and the number of
weak interactions.
24
Sometimes the difference of energy between the native structure and the unfolding protein can be very low:
also in physiological condition the protein can change his 3D structure and this allow to control the function
of the protein.
This is typical of the complex protein, special for proteins with quaternary structure: just a little modification
of a parameter can shift the equilibrium.
Talking about quaternary structure, the weak interactions involved in the folding are the same of the tertiary
structure:
a. Hydrophobic interactions: hydrophobic AAs of one polypeptide chain interact with hydrophobic AAs
of the other one.
b. Hydrophobic bonds: apolar side chains into inner side of proteins, protected by direct contact with
water occur between different polypeptide chain; they can reduce the distance between polypeptide
chains.
c. Electrostatic interactions: occur between ionic or polar AAs of different polypeptide chain.
d. Disulphide bonds are quite strong and are useful to fix the structure of a protein, so the can prevent
the change of conformation into different one.
e. Ion metals (Zinc ions): these kind ions can interact with specific AAs and favour the interaction of
different AAs.
f. Hydrogen bonds.
Folding process 1. Formation of primary structure: transcription of AAs inside the ribosome. As soon as a part of the
linear primary structure is formed, the folding starts.
2. Formation of a secondary structure.
3. AAs starts to interact in order to create the tertiary structure. The
first weak interactions which occurs are hydrophobic one: they
cause the hydrophobic collapse (movement of the hydrophobic AAs
towards the core of the protein, in order to prevent the touch with
aqueous environment).
During the formation of the tertiary structure, chaperons favour the
formation of metastable conformation and reduce the time of the
folding: only the 3D structure characterized of the minimum of
energy.
Moreover, the formation of pro-sequences regulates the folding:
these are the starting 3D conformation favoured by the presence of
chaperons: these can control the folding of other linear sequences
and favour only specific secondary and tertiary structure.
4. Transient intermediate that represents partially folded form.
5. Final modifications (H bonds, elimination of water from hydrophobic core) necessary to generate
folded protein; chaperons allow only the less energized proteins to continue the folding, while the
others are disrupted.
Folding problems
Each AA can exist is 3 different stable structures: this leads to the Levinthal’s paradox: it’s impossible for a
protein to sample all possible conformations. This imply that protein folding is steered by kinetics.
In vitro folding process
Laboratory simulations make the study of weak interactions possible. The first type of weak interaction
studied was the disulphide bridged of the ribonuclease A, which native structure is stabilized by 4 disulphide
bridges, thanks to the presence of 8 cysteine AAs. Only precise positions of disulphide bridges are connected
25
to the activity of the protein. In vitro, it’s possible to denaturate the ribonuclease A using reagents (such as
urea or mercaptoethanol); such agents can reduce the disulphide bridges in order to obtain thiol groups. This
leads to a quite linear protein structure, without loops.
By controlling the oxidation is possible to favour the formation of other disulphide bridges; this allow the
formation of different conformation: native or aberrant, where disulphide bridges are located in different
places. It is now possible to evaluate the protein activity and purify the native protein from the others.
Repeating the same steps in the aberrant conformations is possible to obtain all native conformation.
This happens also in vivo: the native conformation is due to the correct position of specific weak interactions.
In vivo there are specific enzyme able to control the protein folding and to change any aberrant 3D structure:
Protein disulphide isomerase (PDI) can control the position of the disulphide bridges as characterized
by a thiol group: it can localize the inactive fold and change the position of the bridge. This is due to
the presence of a thiol group: it reduce such kind of disulphide bridges by linking itself to the primary
structure realising one thiol group which is very reactive and destabilize the disulphide bridge.
Peptidyl prolyl isomerase (PPI) can confer the correct conformation to proline, which is a cyclic AA
involved in the stability of alpha-helix, conferring rigidity through his trans conformation. If PPI finds
a proline in his cis conformation, it moves the side chains from cis to trans conformation.
PRIONS
Prions are proteins present on the membrane of neurons and which might be useful during the signal
transmission. These physiological proteins may start to fold into aberrant 3D conformation: this could cause
Scrapie (neurodegenerative disease in sheep).
This pathology transforms the secondary structure completely:
the percentage of alpha-helix decreases and is substitutes by
beta-sheet; this causes a different tertiary structure. The
consequence is that the physiological activity changes and the
pathological PRPs form plaques which cause the degeneration
and the death of neurons. The other consequence is that the
pathological PRPs are able to change the conformation of physiological PRPs, which form plaques themselves.
There are different pathologies connected with the formation of pathological PRPs, they all transmit the
Transmissible spongiform encephalopathies (TSEs). The most know TSE is the Bovine spongiform
encephalopathy (BSE). The main issue is that animal PRPs are recognised by human body. Kuru was the first
TSE studied in humans and is located only in Papua Nuova Guinea, where people used to practice
cannibalism.
Other neurodegenerative disease are caused by the possibility of other protein to aggregate: Alzheimer is
caused by deposition in brain tissue of b-amyloid protein.
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Protein study
In order to study a protein (knowledge of primary, secondary and tertiary/quaternary structures), it’s
important to follow different steps:
1. Protein extraction: extraction from cell (in a biological fluid, from an autopsy) and solubilization in a
proper buffer.
a. Protein solubilization;
b. Protein quantification: test of the efficiency of the extraction method.
2. Protein purification, as the spectrometry requires pure and simple samples.
3. Protein sequencing by mass spectrograph.
4. Protein three-dimensional structures: determination of tertiary and quaternary structures
1. Protein extraction 1.a. Protein solubilization The first step is to choose the most efficient solubilising buffer: a solution which is able to solubilize all
proteins present in the cell, tissue or biological fluid. In a cell culture there are different kind of protein:
hydrophobic proteins solubilize in aqueous buffer, but there are also hydrophobic protein, which are
solubilize in organic solvent.
During the extraction it’s important to preserve the protein stability as during this step it could easily be
disrupted. The protein stability depends on:
pH: pH range of stability; a change in pH could cause the denaturation of the structure.
Temperature: in order to reduce protein degradation, the temperature of storage of the solution
must be low (-20° -80°). Moreover, it’s possible to work with specific gasses (nitrogen and argon)
which prevent the contact with oxygen.
Enzymatic digestion: the disruption of the cellular structure implies the release of digestive enzymes
like proteases. In order to avoid the protein modification, every buffer contains enzymes which
inhibit the proteases.
Superficial denaturation: proteins denature in interface air-liquid (oxygen oxidize the protein) or in
contact with glass walls. This phenomen increase with foam formation, which increase the interface
area. Nowadays, specific plastics avoid foam formation.
During the protein extraction it’s possible to use native or denatured conditions: the first possibility is
necessary when the study implies the preservation of the function of the protein, while other analysis don’t
need the native conformation.
Normally the denatured condition is preferred: this allows the use of specific reagents which increase the
number of protein able to solubilize in denaturing buffers. Moreover, denaturing buffers are characterized
by more stable conditions.
1.b. Protein quantification The second step is to demonstrate the extraction conditions: it is necessary to quantify the efficiency of the
extraction method. Assays must be sensible, reproducible and available.
Enzymatic assays Enzymatic assays are the most used and commercialized assays. They use one specific enzyme which is able
to catalyse one specific reaction: an high concentration of one specific product would colour the solution.
They are also defined immunological assays as they use antibodies (small and globular proteins produced by
27
immunological system and able to recognize one/one family of proteins). For this reason the reaction is
proportional to the number of proteins.
RIA (Radio-Immunological Assays) RIA use radioactively treated antibody, so today is not widely used.
ELISA (Enzyme-Linked Immuno-Sorbent Assay) Elisa method is a protein quantification based on the using of two different
antibodies.
i. A solid support contains the primary antibodies, which recognize the
extracted protein;
ii. Incubation of the solid support with the sample;
iii. During the incubation time proteins are directly linked to the primary
antibodies;
iv. Add of secondary antibodies: they recognises the protein linked to the
primary antibody and lead an enzyme (which will catalyse a visible
reaction); there is the creation of a macro-complex.
v. Addition of a specific substrate which is recognized by the enzyme, so
the reaction happens. The intensity of the signal is directly
proportional to the concentration of the extracted protein.
The reaction can lead both to a coloured product or to the presence of
fluorescence.
The Spectrophotometer can measure the absorbance emitted by the products
of the substrate transformation. Its operation consists in applying a specific
wave length to the sample, which absorb it and emits another wave length.
Through the Lambert-Beer law, absorbance correlated to:
The ratio between the intensity of initial (𝐼0) and final (𝐼) wave length;
The concentration of the solution (𝑐);
Two specific parameters which depend on the type of the instrument or the type of sample:
o Optical path length (𝑙) depends on the spectrometer,
o Extinction coefficient (𝜀) (fixed) depends on the type of instrumentation and sample;
Therefore, the measure is proportionated to the amount of proteins, thanks to the Lambert-Beer law.
𝐴 = log (𝐼0
𝐼) = 𝜀𝑐𝑙
2. Protein purification
After having extracted the proteins, it is
necessary to purify the solution as it could be
too complex in order to identify proteins. it is
necessary to dived the initial protein solution
into different solutions, characterized by a
low number of proteins with the same
chemical feature. Depending on the type of
purification method, the subdivision could
be based on different characteristic.
28
2.a Ultracentrifugation The first experimental methodology is the ultracentrifugation ad today is not broadely used. The
ultracentrifugation is able to dived proteins according to their size and density. Different forces are applied
and make protein precipitate according to their MW: low forces make high MW proteins precipitate, while
increasing the force, it is possible to reduce the MW of the precipitate proteins.
Nowadays, it’s useful in order to separate organelles: low forces make nuclei precipitate.
2.b Salting out
Another quite disused method is the salting out: this separate proteins
on the basis of their different solubility; the main issue of this method is
that isn’t a completely reproducible.
Protein solubilization consists in the formation of weak interactions
between proteins and water. Soluble proteins have a thick hydratation
layer around them, but as it’s formed by weak interaction, can change
depending on the solvent polarity, the pH and temperature and the salts
concentration (add of net charge). In this last case, water molecules interact with salts and this makes the
hydratation layer around proteins decreases: proteins aggregate and precipitate.
The most used salt is ammonium sulphate, which is compatible with proteins and change their solubility as
characterized by the presence of both 𝑁𝐻4+ and 𝑆𝑂4
2−. The firsts proteins that precipitate are the
hydrophobic one, as characterized by an already thin hydratation layer.
2.c Chromatography. Today, the most used technology in order to purify proteins solution is the chromatography; there are
different type of chromatographic techniques, but all have quite the same component.
All kind of chromatography consider the interaction between a stationary phase inside a column – which is
a fixed phase that cannot be moved or changed – and a mobile phase – a liquid solution which moves inside
a column –. Stationary phase can be different and functionalized depending on the type of technique.
i. LOADING STEP. The first mobile phase that runs inside the column is useful in order to create
interaction between proteins and functionalized stationary phase.
ii. ELUTION STEP. A second mobile phase disrupts the interactions between proteins and stationary
phase.
The main point is that during the loading sample, there is only one complex
solution formed by an huge number of proteins: all these proteins are able
interact in a different with the stationary phase according to their affinity
with the stationary phase. During the elution step, the movement of a
specific mobile phase able to disrupt specific interaction with stationary
phase, is possible to obtain different solutions: the first protein which exit
from the column are characterized by a low affinity with the stationary
phase as they cross the column quickly (first pick).
29
The process is conducted under high pressure, which allows higher flow rates and minimum separation time
(HPLC: high pressure liquid chromatography)
2.c.i. Size exclusion or gel filtration chromatography. In this technique, the stationary phase is a specific cross-
linking polymer which produces a matrix: proteins are
separated according to their molecular weight. The matrix is
characterised by a specific pores dimension: protein with an
high molecular size cannot enter in the pores and interact
with the surface and this decrease their velocity to cross the
column. Therefore, the first section has an high molecular
size, which decrease with the further mobile phase. The
interaction within protein and stationary phase is a physic
bond.
Nowadays this method is mainly used for polymers and not
for proteins as isn’t completely reproducible; still is usable to
purify solution from the reagents used in the extraction step (low MW).
2.c.ii. Ion (cationic or ionic) exchange chromatography. The ion exchange chromatography is able to separate proteins
according to they charge: the presence of acid or basic AAs. This
technique allows the division between anionic and cationic proteins:
anion proteins are normally characterized by a pI lower than 7, which
means that they contain more acidic AAs then basic AAS (negative
charge), cationic proteins are normally characterized by a pI higher
than 7, which means that they contain more basic AAs then acidic AAS
(positive charge).
The stationary phase is charged: this allows to talk about:
ionic exchange chromatography: positively charged stationary
phase creates strong bound with anionic proteins, due to
amino groups;
cationic exchange chromatography: negatively charged
stationary phase creates strong bound with cationic proteins,
due to carboxyl groups.
The experimental protocol implies e.g. a negatively charged stationary phase (cationic exchange
chromatography), which could bound cationic proteins.
The first mobile phase must preserve the charge of the proteins, so the condition of the loading step are
physiological; this also allows to maintain low the salt concentration, which could compete with proteins to
interact with stationary phase.
During this step, anionic proteins feel a repulsion toward the stationary phase: this allows them to quickly
move through the column.
Successively, in the elution step, the mobile phase is changed: the increase of salt concentration increases
the ionic strength in the solution. The competition between salts and proteins allows to separate different
solutions.
30
2.c.iii. Hydrophobic interaction chromatography (HIC) This technique separates proteins according to their hydrophobicity. The stationary phase is hydrophobic, so
it is able to perform hydrophobic interaction with hydrophobic AAs.
In order to favour the hydrophobic interactions between the core of protein and the stationary phase,
hydrophobic AAs have to be moved towards the surface by the addiction of salt. This increases the
concentration of ions whom interaction with water reduce the interaction between water and the
hydratation layer: hydrophobic AAs move towards the interface.
In the elution steps the concentration of salt is reduced. Removing ions, water molecules creates a new
hydratation layer and move the hydrophobic AAs to the core: this disrupt the hydrophobic interactions with
the stationary phase.
2.c.iv. Reverse phase chromatography (RPC). This technique, is based on the same rudiments and separates
protein according to their hydrophobicity. It’s more efficient than
the HIC, so is more used. The stationary phase is an hydrophobic
matrix which is able to perform hydrophobic interactions with
proteins. The stationary phase is characterized by the presence of
arms formed by an high number of carbon atoms. Their length is
useful to directly interact with the core of proteins. There isn’t the
increase of salt during the elution steps. The loading step is
performed in aqueous solution, without salts.
The elution step is performed changing the mobile phase from
acquit to organic: the increasing of the concentration organic
solvent represents the competitor for the proteins as it interacts with the e stationary phase. the tech is
called reverse because it’s perform in aqueous solution and the concentration of organic solvent in the
organic solvent is increased.
2.c.v. Affinity chromatography This technique characterized by a very specific stationary phase: it’s functionalized
with a specific reagent or biomolecule. The functionalization is useful to perform
one specific interaction with one specific protein.
it useful to select one specific protein (or one family) in the sample.
For example in the neuro-affinity chromatography, the stationary is linked to an
antibody : during the loading step only protein recognised by the antibody link to it.
The elution step aims to disrupt the specific interaction; this could be by changing
the experimental condition (pH, T) or by the addition of a competitor (same or
similar protein in high concentration).
2.d. Poly-Acrylamide Gel Electrophoresis (PAGE) 2.d.i SDS-PAGE Electrophoresis means the possibility to separate proteins according to their MW when a magnetic field is
applied. This is possible thanks to a specific matrix in polyacrylamide gel.
The first step is to create the gel through the polymerisation of a liquid solution containing:
2 monomers: acrylamide and bisacrylamide ;
A catalyst: TEMED (tetramethylethylendiamine). The reaction would occur also at low temperature,
but the catalyst reduces its time.
An initiator: APS, which starts the reaction.
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NATIVE ELETROPHORESIS. This kind of PAGE uses proteins in their native state, which are separated thanks
to their own charge. This technique is not wide used as AAs often interacts each other, reducing proteins net
charge: the migration would be very slow and different proteins could not well separate.
SDS-PAGE. For this reason, the denaturated technique is often used:
a reagent (DTT, b-mercaptoethanol) cuts the disulphide bridges and disrupt the tertiary structure.
The second reagent is the SDS; this is an amphipathic molecule due to its long carbon chain and the
phosphate group. The carbon chain interacts with the hydrophobic or non-polar parts of the
polypeptide while the phosphate confers negative charge to the proteins.
Eventually, the SDS can confer a linear conformation to the protein and increase the number of
negative charges. This last point is important to improve the mobilization inside the matrix.
The rule of the separation is the possibility
of proteins to enter inside the pores of the
gel: high MW proteins remain on the top of
the gel as they cannot entre inside the
pores, while only very small proteins are
able to cross all the gel matrix.
The migration occurs under the influence of
electric field and towards the positive pole
(down). As all proteins are negatively
charged, the charge density is comparable
to their MW.
The migration is visible thanks to the addiction of a blue colorant: this is a small molecule which runs faster
than proteins. When the blue colorant is on the bottom of the gel, all proteins are inside the gel.
After the electrophoresis the gel is colourless, therefore it is put inside a semi-solutions in order to visualize
the separation better. It is possible to choose between different semi-solution according to the protein
concentration:
Normally the the Coomassie Brilliant Blue is used: it can detect a quantity of protein about 50ng
protein.
If the sample is characterized by a lower proteins concentration, it is possible to used the Silver
Nitrate (dark) which can detect a protein quantity of 1 ng.
The protein migration depends on different experimental condition:
Temperature (normally room temperature). If there are thermolabile protein is possibl to perform
it at low temperature.
Voltage: intensity of the electrical field. If it’s higher the time is reduced. Anyway, in the first step
this can’t be too high because the solution is too complex: the voltage is reduce in order to favour
proteins to enter inside. Later, the high MW proteins have stopped in the first part so the protein
sample has a lower number of proteins.
Monomers concentration: proportional to matrix pores. In order to separate a low MW proteins
solution, it’s possible to increase the monomer concentration: the efficiency is increased.
WESTERN BLOTTING
The western-blotting is another technique, which can identify one specific proteins and is usually performed
after the SDS PAGE.
32
i. Proteins are transferred on a specific membrane
(nitrocellulose or PPDS) through an electric field: negatively
charged proteins link to the PPDS through contact.
ii. The membrane could be incubated with a specific antibody
(primary) solution, able to bind a specific protein present
in the gel.
iii. After the incubation, the same membrane is incubated
with a solution with the secondary antibody. Its function is
double: first of all it has to recognize the primary antibody
and to link to a specific enzyme (which recognize a
substrate) able to catalyse a coloured or fluorescent
reaction.
This technique is often used in diagnostic (e.g. prion pathological protein).
2.d.ii 2D electrophoresis The 2D electrophoresis can perform a double separation: one according to the pI and another according to
the MW.
The first separation is performed inside a polyacrylamide gel polymerized on
a linear plastic support: the gel is characterized by a low concentration of
monomers, so the pores are huge. Indeed, the pores dimension in the first
separation is not important as the MW can be neglected. The main goal of
initial step is to favour the mobilization of protein through all the stripe and
separate proteins according to the pI (acidic or basic AAs concentration). in
order to do this, the gel is characterized by the presence of immobiline, which
are characterized by a negative or positive charge. The presence of different
immobile inside the gel Is used to form a gradient of pH (starting from a very
low pH condition and increase it up to 10 o 11).
Therefore, proteins migrate inside the
gel: when they found themselves in a
region characterized by a pH similar to
their pI, they stop.
In the end, after having loaded a complex
solution, the first separation leads to a gel with different stripes containing
same pI proteins. Firsts bends are acidic, lasts basic.
The second separation occurs according to the MW. In this case the strips are physically loaded on a
polyacrylamide gel. the same of the. The process is similar to the one-dimensional electrophoresis, expect
that in this case there isn’t one complex solution, but different proteins bends. After the application of a field,
proteins migrate inside the gel thanks to the SDS. At the end of the process, it is not possible to recognise
bends, but proteins spots, due to the double separation.
The 2D electrophoresis has an higher efficiency compared to the 1D electrophoresis as each spot is normally
due to one single protein.
In the end, it is possible to use specific software, able to compare different 2D electrophoresis results and
detect the differences in spots: this allows to identify possible pathological biomolecules.
Such kind or technique is useful to proteomics, which is a scientific field whom aim is the identification of
proteins.
33
3. Protein sequencing The most used analytic technique in order to sequence a protein is the mass spectrometry. While it took 10
years and 100g of insulin to Sanger to sequence it, nowadays it’s possible to identify huge proteins in just a
couple days using few µm of it using the spectrometry mass.
Identifying the primary structure is important because the primary structure controls all other structure, so
this step is the beginning of the proteomic characterization. Moreover, pathological proteins could be
identified by a few different AAs, so it is useful to know the whole AA sequence.
3.1. Sequencing preliminary steps
3.1.i N-terminus analysis In the past the first step was to identify the terminus in
order to know the number of polypeptide chains. It
was used the Dansyl chloride is a fluorescent reagent
which links to the ammine groups present is the N
terminus in basic environment. After this interaction,
they suddenly change the environment from basic to
acid and the low pH allow the hydrolysis of all peptide
molecule. This release all AAs and the N-terminus bind
to the Dansyl chloride: they are characterized by an
higher MW and fluorescent. It allows to quantify and
identify the N-terminus AAs.
Nowadays, MS is an high resolution instruments which
quickly recognises the terminus, so the Dansyl step is
not used nowadays.
3.1.ii Breaking disulphide bonds On the contrary, the way to disrupt the disulphide bonds is the same of
the past. These are broken using DDT (β-mercaptethanol), which reduces
the bridges to thiol group and favour the denaturation of the protein.
In MS protocol the reduction is followed by the alkylation step, which
consists in the addition of iodoacetamide (IAA) which prevent the
formation of new disulphide bridges.
3.2. Polypeptide fragmentation The polypeptide fragmentation is necessary in order to reduce the protein size before the injection. It the
past it was performed the chemical fragmentation using the Cyanogen bromide (CNBr) which recognise the
methionines present inside the protein, link to these and cause the disruption of the polypeptide.
Enzymatic fragmentation This reaction is not very efficient, so nowadays the enzyme fragmentation it is performed through an enzyme
which recognise more than one specific peptide bond. The most common used is trypsin: it is able to
recognise each peptide bond between 1 arginine and 1 lysine and hydrolyse it. It’s high specific and high
efficiency: each peptide bond between arginine and lysine is digest by it. As these bonds are very common,
we can assure that the polypeptide chain is reduced into an high number of short peptide.
Other possible enzymes are chimotrypsin, elastase and pepsin, all physiologically produced in stomach and
pancreas with the function of digesting proteins. Nevertheless, chimotrypsin, elastase and pepsin are not
34
broadly used as they have a not high specificity, so the peptide length could be too high for the mass
spectrometry.
Moreover, trypsin is able to digest protein inside an heterogenous phase (the polyacrylamide gel), therefore
the procedure is considerably simplified: it is possible to physically cut the bend, perform fragmentation and
incubate the gel with trypsin solution overnight. At the end a solution formed by an high number of digested
peptide is obtained.
3.3. Peptides identification The final step is the identification of all AA present inside the peptide.
3.3.a Edman degradation In the past the process was chemical, using a reagent
(Edman reagent) which can link to the N-terminus
present in each peptide chain. The interaction is
performed in basic environment an changing
environmental condition, the hydrolysation occurs
realising the AA linked to the reagent. This process
separates the AAs from the peptide which can be
identified by chromatography. The analyses time is high
as the number of reaction is proportioned to the
number of AAS.
3.3.b Mass spectrometry (MS) Today, the peptide solution can be inject in the mass spectrometry which identifies the AAs in more or less
1 hour. This instrument is able to measure the ratio between m (mass) and z (charge) of ions present in gas
phase: the first step is to create ions in gas phase starting from a liquid solution.
35
The first part of the MS is the source
i. Peptides digested by trypsin are dissolved in a polar and volatile solvent.
ii. It occurs the vaporization of sample by high pressure, as the solution is injected through a
capillary. An high voltage disperse samples into an aerosol of highly charged peptides: the electric
field take off the surface electrons, so the peptides get positive charged.
iii. Charged peptides are reduced in size by the solvent evaporation, assisted by a warm flow of
nitrogen.
iv. The second step is the analyser, which allows to
separate ionized peptides according to their charge-
mass ratio: charged particles are affected by an
electromagnetic field and produce a trajectory
depending on m/z.
v. The analyser consists in electrodes which produce
magnetic field: only ions with specific m/z can cross the
whole field and exit to be registered by the detector. The
field changes in function with time, so all ionized peptides
can eventually be detected and produce a peak.
vi. Then, specific software analyse the graphs. In order to
have an high resolution, the picks width should be small,
because this means the peptide have been separated
according to a small difference between the ratio.
Another important parameter is
the transmission: the ratio
between the number of ions
generated in the source and the
number of ions arrived to the
detector.
Hereafter, specific databases can be downloaded in order to compare the spectra obtained to all spectra
referred to a group of proteins through specific software. This allows to create a list of all proteins present
in the sample.
36
Protein database The most widely known database are:
SWISS_PROT is a database of annotated protein sequences; it also contains additional information
about function of the protein, their domain structure, post-translational modification(s), etc.;
o TrEMBL is a supplement to SWISS_PROT, which contains all protein sequences, translated
from nucleotide sequences of the EMBL database.
NCBInr (National Center of Biotechnological Information) is a database containing sequences
translated from DNA sequences of GenBank and also protein sequences from other databases like
SWISS_PROT;
Talking about UniProt, the possibility to download the specific proteins database is performed by the
specific taxonomy of the sample (human culture cell or other species cells). After having choosing the
correct taxonomy, there is the possibility to download 2 different database: the reviewed or unreviewed
one.
When it is necessary to identify protein of an unknow sample (where the species is not present in
the bank), then analysis is manual: the MS spectra are compered to database data referred to the
major family of the species: it possible to obtain a long list of proteins.
This comparison allows to obtain a long list of unknow peptides: peptides non present in well-
known proteins, but identified in the sample.
The second step is to look up for the same peptides in specific other software (BLAST) to know the
name of the proteins present in other species with an high coverage (the most similar to the
peptide in the database). Consecutively, we know the all peptide could be referred to homology
proteins and allows to selects the MS spectra referred to homology proteins.
These are referred to unreviewed proteins database.
On the contrary the reviewed database contains all MS spectra refereed to specific proteins, after
the experimental validation.
NCBI is a very large database with peptides, DNA and RNA sequences. The specific feature of this platform
is the possibility to download the dbEST database containing all the sequenced mRNA. This is useful
working with unknow species (or with uncomplete genome sequence): the translation of unknow peptides
in mRNAs gives further information about the species.
This database increases the number of identified peptides: when the match between experimental mRNA
and the database is high, the database give back the name of all possible protein.
In this case the identification is not so efficient but increases the number of information.
Database-searching programs There are several dataset-searching programs available over the internet, characterized by different
algorithms useful to identify proteins like MASCOT and SEQUES. They differ according to the type and the
resolution of the MS.
In conclusion, there are three main technique of proteins sequencing. The MS analysis is useful to identify
primary structure and consecutively it allows to identify proteins. Nowadays is often used in proteins
quantification inside cells.
Moreover, X-ray and NMR allow to obtain information about secondary e tertiary structure.
37
Protein classification Proteins can be classified according to different parameters.
Considering the 3D structure it is possible to recognize:
Membrane proteins: interact with cellular membrane (high hydrophobicity)
Globular proteins: globe-like shape e.g. haemoglobin;
Fibrous proteins: long and thin structure e.g. cheratin, collagen
Fibrous protein These form the simplest class because they are all characterized by a simple primary structure with a low
number of AAs. Moreover they have a regular secondary structure (alpha or beta). This is involved in the final
mechanical feature: they have an high strength, an high resistance (their main function is the protection) and
an high flexibility. Their final structure is filamentous.
They are present in cell walls and in connective tissue, like tendons and ligaments: they confer strength and
rigidity to the structures and physically hold them together.
The main fibrous proteins are α keratins (hair), silk fibroin (used in medicine), human collagen. In these
proteins, cysteine is present in high concentration (mainly in keratins) as it’s involved in the formation of
specific tertiary structure. Proline is present mainly in collagen because it’s involved in its stabilisation.
KERATINS There are two major groups of keratin with similar structure: α-keratins (in mammals) and β-keratins (in birds
and reptiles).
This proteins is the component of hair and nails.
It confers strength, un-reactivity and resistance thanks to its secondary and
tertiary structure. It is formed by 2 polypeptide chains able to obtain a single
α helix: 2 α helices interact each other with H bonding in order to form a
super helix.
α helices interact each other through hydrophobic interaction: this stabilize the hydrophobic AAs in the core
of the structure and favour the formation of the super helix. On the contrary, hydrophilic AAs are in external
positions.
Successively, the super helix is obtained thanks to the presence of cysteine: their high number formed a lot
of disulphide bridges which reduce the dimension of the super helix and increase its mechanical resistance.
In conclusion, the unit of keratin is the super helix, whom high strength is due to a precise organization of
super helices.
There are 2 types of keratin which interact each other in order to
form 1 heterodimer thanks to the presence of specific hydrophilic
AAs. The acidic super helix (type I) have an higher concentration of
acidic AAs (negatively charged), while the type II (basic super helix)
has more basic AAs (positively charged). This allows one acidic
superhelix to interact with 1 basic super helix due to the attraction
between different charges.
Moreover, more heterodimers interact each other in order to form
one protofilament.
More protofilament can interact each other to form one protofibril.
3 protofibril interact each other to form a microfibril.
More microfibril interact each other to form a macrofibril.
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Obviously, the primary structure is involved in the final organization into macrofibril: one single mutation
present in t eh AAs could be involved in pathologies because of the lost of organization. This cause disorders
to skin integrity, cell adhesion, motility, proliferation.
SILK FIBROIN CLASS Silk fibroin is produced by silkworms (Bombyx Mori’s webs) and spiders.
The primary structure is quite simple and consists in the repetition of only 3 AAs: Gly-
Ser-Ala. Glycine confers flexibility to the structure and this latter increases the
number of peptide which can interact each other. As also serine and alanine have a
small sterical hindrance, in the unit of volume there is an huge number of polypeptide
chains.
Another feature is the position of different polypeptide chains: same residues interact
each other and found themselves close; this allow to reduce the space between 2
polypeptide chains and increase their number in a volume unit.
This primary structure favours the formation of beta sheets. A zig zag
conformation is formed thanks to H bonding between different AAs.
Silk proteins are similar to the keratins one: it is characterized by an huge
strength due to the strength and the number of chemical binding between
different AAs.
Moreover, the silk fibre has resilience and flexibility; this allows the protein
to change its shape by the application of a force an to recover its initial
shape after the release. This feature is possible thanks to the H bound which can be disrupted and formed
again in the same positions.
SPIDER SILK The silk produced by spider is the one of their webs. Spider fibres have a similar structure to the silkworm,
but has a major number of glycines. The consequence is that the fibre can increase the number of polypeptide
chains in a volume unit because its sterical hindrance is minor. This causes an higher fibre strength.
The other important feature is that this structure is compatible with other different compounds (sugar, ions)
which increase its application in medicine. Spider silk is used in tissue regeneration: formation of a scaffold,
grown factor….
Other application are in optical instruments thanks to its very efficient signal transport.
COLLAGEN (human) Collagen is the major component of connective tissue and in particular of skin, tendons, ligaments, teeth and
bones.
Its unit is a triple helix (each polypeptide chain is an alpha helix).
There are 30 type of collagen due to the high number of genes which produce it. These type are divide in 4
major classes. In particular, type 1 is formed by 2 identical triple helix while number 2 has 3 identical
polypeptide. Type 3 is the principal wall components of arteries, intestine and uterus; type 4 (forms epithelia,
wall of blood capillaries and kidney’s glomeruli .
The general structure of collagen is the following.
The primary strucutre is the simple sequence of Gly – X -Y , with an high concentration of glycine which
stabilize the secondary structure. X is often proline and Y lysin. These latter could be hydroxylate (bind to an
hydroxyl group) to favour the formation of specific weak interaction between different polypeptide chains.
39
The proline is characterize by a cycle (high sterical hindrance): it confers rigidity to the structure as it prevents
any movement. This cause a quite fixed secondary and tertiary structure.
This primary structure favour a specific secondary structure: the formation of an α helix. Proline
will always be on the external in order destabilize less the structure and in contact with the
external environment, while glycine will be closest to the helix axis in order to interact with other
α helix.
This interaction occurs between three different polypeptide chains
and it’s through hydrogen bindings performed by the H of glycine
and O of carbonyl group of proline.
Different triple helix are able to interact each other through weak interaction
(van der Walla, hydrogen bonding) and create a compact and resistant
structure.
In the quaternary structure also occur intra molecular cross linking within
individuals helices due to the H bonding between Gly and Pro. On the contrary
the inter molecular cross linking is due to the interaction between different triple helix.
BYOSYNHTESIS OF COLLAGEN 1. The collagen synthesis starts in the ribosome with the production of the preprocollagen (primary
structure, polypeptide chain).
2. This is formed also by a little sequences of AAs (signal sequence) not present in the final collagen,
useful to control the transport inside the endoplasmic reticulum. It is characterised by the presence
of a C and an N terminal.
Here a lot of reaction occurs: preprocollage is functionalized to the correct form.
3. The first step is the hydroxylation of selected proline and lysins. This is important because it allows
the interaction between 3 different polypeptide chains in order to form the H bonding inside the
triple helix.
4. The second step is the binding of specific oligosaccharide in the C terminal. These are useful in order
to control the final interaction of 3 different polypeptide chains.
5. Initial glycolisilation of hydroxylysine residues.
6. After this, disulphide bridges are formed in the C terminal: they start the arrangement of collagen
molecule. They move 3 different polypeptide chains close each other in order to favour the formation
of hydrogen bonding.
7. Formation of the procollagen.
8. After this, the initial triple helix is transport into the Golgi system inside a vescicle.
9. Exocytosis transfer triple helix to extracellular matrix.
10. Removal of the of the additional C and N terminal: this is called tropocollagen.
11. Different tropocollagen formed by triple helix interact each other in order to form intermolecular
cross linking and obtain the final fibrils.
40
Collagen is very susceptible to mutations and dysfunctions, one well know is the osteogenesis imperfect. It’s
referred to type 1 collage which has a single mutation: all glycine are substitute with cysteine or asp or
aspartame. This cause a defective folding of collage helix: this lost in resistance and flexibility. It’s correlated
with problem in connective tissue and bones growing.
Osteoporosis affects an high concentration of old people and is caused by a low produce of collagen. The
treatment consist in the possibility to increase the collagen production with grow factors.
Scurvy is caused by deficiency of vitamin C or ascorbic acid. This is important in the synthesis of collagen as
it’s a coenzyme involved in the hydroxylation of collagen. It cause the production of a low resistance collagen.
Collagen is used in medicine as it’s an endogenous tissue characterized by resistance and flexibility: valves
and other system are made in collagen in order to favour tissue regeneration. As it’s a self material, the
immune response is completely stopped.
It also can be used as a drug deliver.
Muscle proteins Muscle contraction is due to the interaction between specific fibrous proteins. Muscle itself is constituted
by various proteins and other organelles.
Muscles system’s function are to permit movements, maintain standing position and permit life,
transforming chemical energy into movement.
Muscles are a soft tissue, containing protein filaments of actin and myosin able to produce contraction that
changes both the length and the shape of cell.
It is divided into three types:
Skeletal muscle or “voluntary muscle”: is anchored by tendons to bone and is used to move and to
keep the posture,
Smooth muscle or “involuntary muscle”: is not under conscious control and is found within the wall
of internal organs,
Cardiac muscle or myocardium: is involuntary and only present in the heart. It has a similar
structure to skeletal muscle.
41
Voluntary muscle Voluntary muscle is involved in movements and in the possibility to maintain position in the space: it is able
to control muscle contraction.
It’s connected to the bones through tendons
and it has a precise organization. Is formed
by an high number of different fasciculi, each
formed by an high number of muscle fibres,
formed by myofibril, formed by sarcomeri.
The sarcomere is the unit of the skeleton
muscle.
SARCOMERE
Each sarcomere gas 2 mobile extreme, the Z
disk.
They are formed by actin myofilament (thin
filament) connect to Z disk and myosin
myofilament (thick filament). Contraction is
due to the movement of actin myofilament
onto myosin myofilament in order to reduce
the length of the sarcomere or increase it
(during the relaxing time).
MYOSINE Myosin is an elongated fibrous protein, characterized by a double alpha helix. The secondary structure is
similar to the one of the keratin: a double alpha helix.
One molecule is formed by 6 polypeptide chain: 2 are define as heavy chains, other 4as light chains: among
this, 2 are useful to bind actin, the other 2 are involved in regulation for myosin contraction.
Myosin could be divided into 3 different domain:
a. The head domain binds the filamentous actin and uses ATP hydrolysis to generate force and to
"walk" along the filament
b. The neck domain acts as a linker and as a lever arm for transducing force generated by the catalytic
motor domain. It’s also a binding site for myosin light chains which generally have regulatory
functions
c. The tail domain generally mediates interaction with other myosin subunits. It may play a role in
regulating motor activity.
42
ACTINE The unit of actin thin myofilament is the G-actin, a globular
protein. Different G actins interact each other in order to
form dimers, trimers and very long filaments (F actin). This
is defined as a fibrous protein due to the huge number of
interaction between G actins.
From a functional point of view, F actin is characterized by
one sized involved in the binding with the Z disk and a lot
of binding sites involving ATP or specific ions (𝐶𝑎2+ and
𝑀𝑔2+).
There are other 2 important proteins involved in contraction regulation: tropomyosin and troponin.
TROPOMYOSIN is fibrous protein able to interact with actin myofilament. Its function is to bind and
hide 7 actin side in order to prevent the bind between myosin’s head and actin and prevent
contraction.
His structure is a homodimer formed by 2 a helices subunits.
Tropomyosin characterize the rest condition and its position is controlled by tropomyosin.
TROPONIN has 3 active sites: one for tropomyosin, one for the 4 𝐶𝑎2+ and the third for actin.
The interaction between calcium ions and troponin is one the first step in otder to obtain muscles
contraction.
Contraction mechanism Muscle contraction is controlled by the nervous system.
i. It starts after a nervous input: at the
end of the neurons there are a lot of
vesicle able to release
neurotransmitters (acetylcholine);
ii. this bind to specific receptors on the
surface of muscle tissue (it. placca
neuromuscolare). This binding is
able to introduce the acetylcholine
inside the muscle because the
acetylcholine is able to be
transferred till the RE.
iii. Here, specific receptors allows the
release of calcium ions inside
endoplasmic reticulum in the
muscular tissue.
iv. Calcium ions interact with troponin:
4 calcium ions bind 1 troponin;
v. this change its conformation and prevent the binding with tropomyosin, therefore, tropomyosin it’s
able to move along the actin myofilament.
vi. This movement releases the actin molecules and favour the interaction between actin myofilament
and myosin heads.
43
Muscle contraction
The muscle contraction is due to the movement of myosin across the actin myofilament. This process
requires energy, which is provided by the use of ATP (interacts with myosin heads).
After the interaction between ATP and myosin heads, the latter change conformation and then the
hydrolysis of ATP occurs. This releases chemical energy, used by the myosin head in order to move along
the actin myofilament. After this the ATP is removed and the cycle start again.
The contraction itself occurs inside the sarcomere. The myosin myofilament move across the actin
myofilament and reduce the sarcomere length by reducing the distance between 2 Z disk.
This is the reason why the muscle is so accurately organized: only a huge number of sarcomere could
permit the muscle contraction.
The ATP molecule provides chemical energy.
At the centre is a sugar molecule, ribose (the same sugar that forms the basis of RNA). Attached to one side
of this is a base (a group consisting of linked rings of carbon and nitrogen atoms); in this case the base is
adenine. The other side of the sugar is attached to a string of phosphate groups. These phosphates are the
key to the activity of ATP and consist in the variable part.
The transformation from ATP to ADP is through the release of 1 phosphate group: the breaking of this high
energy chemical bond release a lot of energy.
44
Globular proteins Globular proteins are characterize by a spherical shape. They can easily interact with external environment
thanks to the presence, on the surface, of many hydrophilic AAs able to interact with a polar environment.
In the core there are all the hydrophobic AAS.
The main functions of globular proteins are:
Enzymes: catalysis of organic reactions inside organisms;
Messengers: transmission of messages to regulate biological processes (hormones, insulin);
Transporters: transport of molecules (e.g. O2);
Structural proteins: globular and soluble monomers which polymerize to form long and stiff fibers
(actin).
IMMUNOGLOBULIN Immunoglobulins are produced by the immune system and their function is to recognize antigens: non-safe
molecules (bacteria, virus, non-physiological biomolecules like aberrant proteins).
There are 5 main immunoglobulins classes:
a. IgG, IgE and IgD are present in blood;
b. IgM, IgA can exists with different molecular weight (monomers, dimers); some of them are present
onto the surface of specific cell of the immune system in order to react with antigens: T or B
lymphocytes, which function is to destroy antigens.
T lymphocytes are produced by the thymus gland and recognize specific antigens and assist to their
destruction.
B lymphocytes are produced by the bone marrow and are activated by antigens to produce and
secrete antibodies binding directly antigens (memory cells) and acting as marker for macrophages.
Immunoglobulin structure (IgG) Ig are small globular proteins, with 2 heavy and 2 light chains. All these 4 chains are connected each other
through disulphide bridges in order to favour the specific 3D conformation.
45
Moreover, there are two type of domains: constant and variables.
The first is the same in all immunoglobulins of the same family,
the latter is different by considering different type of IgG. This
increases the efficiency and the globality in recognising and bind.
The interaction is weak and depends on the structure of IgG and
antigens.
From a structural point of view, the polypeptide chain is organized
in the following way: all polypeptide chains interact each other in
order to form a specific secondary structure, a beta sheet. They
also interact in order to obtain the final spherical 3D
conformation.
Moreover, the final
part to the variable
region is the hypervariable region, which changes also inside
the same IgG family. This is increases the efficiency in the
recognition.
From a functional point of view, immunoglobulins can be
distinguish in 2
different domain:
the antigen
binding fragment (Fab) and the terminus fragment.
One molecule on immunoglobulins is able to interact with 2
molecule of antigens through weak interactions. The interaction
aren’t strong because immunoglobulins function is only to show
the presence of the antibody and not its destructions (which
relies on macrophages etc).
MYOGLOBIN Myoglobin’s function is to transport gas phase molecules (oxygen and carbon dioxide) from blood flux into
lungs and vice versa.
In all vertebrates, there are 2 different carrier proteins: haemoglobin in blood and myoglobin in muscle.
The presence of 2 different proteins is because the efficiency in binding with oxygen is different: myoglobin
is characterized by an higher efficiency of haemoglobin as muscle tissue requires constant high energy.
Myoglobin is a quiet small protein divided into 8 different alpha helix domains, able to interact each other
in order to form an inner core containing the heme group, which bind oxygen.
The heme group is formed by 4 cycle (pyrol) and it’s planar. It can bind 1 iron
atom, which is the atom involved in the interaction with oxygen.
It is also stabilised by 1 histidine present in the seventh alpha helix (through an
H bonding).
The iron bind to the heme group could be present in 2 different ionization state:
𝐹𝑒2+ or 𝐹𝑒3+. Only when iron is present in the feorus state (𝐹𝑒2+) it can bind
the oxygen. When oxygen it’s bond, the colour of myoglobin is red. On the
contrary, when O is realsixe, iron change state and myogobuline changes shade
of color.
The heme core is protected by all alpha helix.
46
The importance of myoglobin is clear considering the affinity of myoglobin with oxygen. The graph of
saturation of myoglobin is called oxygen-binding curve.
On the x axis there’s the pressure of oxygen (similar to
concentration), while on the y axis there is the percentage of
O bound to myoglobin.
The hyperbolic behaviour is due to the favourable
interaction between myoglobin and oxygen. As the high
slope shows, even when O pressure is low, there is an high
concentration of O bound to the myoglobin. An high number
of bonds are present even at 2,8 torr of oxygen, when the O
pressure in venous blood in physiologically 30torr: this
means that also in venous blood the myoglobin is saturated
and that in every condition muscle can obtain the maximum oxygen. Moreover, the increase of saturation
between venous and arteriosus blood is not very high.
Theoretically, muscle tissue can start contraction in every condition, indeed at 𝑃50 = 2,8𝑡𝑜𝑟𝑟 (it means that
at 2,8 torr, 50% of myoglobin in saturated) is very low compared to the O pressure in venous blood
(𝑝𝑣𝑒𝑛𝑜𝑢𝑠 = 30 𝑡𝑜𝑟𝑟, 𝑝𝑎𝑟𝑡𝑒𝑟𝑖𝑎𝑙 = 100𝑡𝑜𝑟𝑟).
HAEMOGLOBIN
Haemoglobin and myoglobin are different from a primary structure point of view, but they derive from the
same ancestor protein. This is expressed in their tertiary structure, which is very similar.
Also in haemoglobin AAs can interact each other in order to form 8 different alpha helix domains in which
core the emo group is bind. This is strictly connected with their biological function: transport O.
Haemoglobin is quiet complex: it has a quaternary
structure. It’ formed by 4 different polypeptide
chains (while in myoglobin there is only one
polypeptide chain).
Of the 4 chains, 2 are called alpha chains and 2 beta
chains. Both are organized into alpha helix domains
which arrange in the shown way.
The difference between alpha and beta is in a few
number of AAs so both has a similar tertiary
structure: both can bind in their central part 1 heme
group. This means that in haemoglobin there are 4 hemo groups in the core of each polypeptide chain.
Hence, the first difference between haemoglobin and myoglobin if the number of oxygen molecules they
can bind: the first saturates with 4 oxygen molecules, the latter with 1 oxygen molecules.
All 4 polypeptide chain are ale to interact each other though hydrophobic interactions or H bindings. These
weak interactions perform the globular shape of haemoglobin.
The reason why there are two different proteins with the same function (transport oxygen) is that the
affinity of haemoglobin for the oxygen is quiet lower: while myoglobin saturates in every blood condition
(considering all possible O pressure: both in arteriosus and venous blood), for haemoglobin the complete
saturation is only possible when in arteriosus blood, when oxygen pressure is very high. In tissues, where
oxygen pressure is quite low, the hemo group doesn’t completely saturates and releases the molecule in
order to oxygenate cells.
47
In conclusion, haemoglobin and myoglobin aim is completely different: haemoglobin must releases oxygen
in tissues, while myoglobin represent a storage of oxygen for muscle, as they require an high content of
energy (oxygen) during their all activity.
Mechanism of oxygenation The possibility to bind or release oxygen is related to the final haemoglobin 3D conformation: all 4
polypeptide chains can exist in 2 stable conformation: T or R. Only the R state is characterized by an high
affinity for oxygen, while the T state prevents such interaction.
Inside the structure of 1 polypeptide chain, after the interaction with the oxygen, there is a little movement
between the hemo group and the alpha helix: the hemo group get closer to the helix. This causes a general
movement of all 8 alpha helices as they all
interact each other.
The thing that mainly change is the
dimension of the central cavity: in T state
there is a quite large central cavity, while
in the R state all polypeptides chains
move and reduce the dimension of such
cavity.
The presence of O causes a general movement of
alpha helix and this disrupt the weak interactions
between alpha and beta polypeptides chains: this
causes the different final conformation.
T state is characterized by ionic interactions
between one aspartic acid in beta chain and one
tyrosine in alpha chain, stabilized by the presence
of another histidine in the beta chain. After the
movement caused by the interaction with the oxygen, the polypeptide chain moves and ionic interactions
are disrupted; nevertheless, new ionic interactions are formed between other AAs.
Hence, both T and R states are stable as chains can interact with the same type of interaction, but they
differ due to the different AAs involved. This causes a reduction in size of the cavity.
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Cooperative binding haemoglobin-oxygen
Inside the haemoglobin, all 4 polypeptide chains bind an
oxygen molecule; the limiting step is due to the first
interaction with the first O molecule. This means that the first
binding between heme group and oxygen is quite low, because
it destabilizes the T states (so the first affinity of haemoglobin
for O is low). Considering the oxygen binding curve, at very low
oxygen pressure the percentage of oxygenated haemoglobin is
low because the first interaction isn’t favoured from a
thermodynamic point of view. Nevertheless, after the binding
of the first O, the polypeptide chain turns into R state and this
favour the movement of the nearest polypeptide chain. This
feature is called a cooperativity phenomenon: the first bind to oxygen catalyse the movement of other
polypeptide chains. In fact, when the oxygen pressure is quite high, the affinity increase quickly up to the
asymptotic value (S-shaped curve).
This means that in venous condition, only a little part of the haemoglobin can bind oxygen molecules, while
in arteriosus blood, this bond in highly favoured.
Graphically, the difference between haemoglobin and myoglobin is clear considering low oxygen pressure.
Allosteric regulation
The central cavity has a central role in the interaction’s regulation. It has the possibility to bind to specific
molecule, called allosteric modulator; this is a biomolecule completely different from the substrate
(oxygen) able to bind with protein is a site completely different from the binding site of the substrate.
In general, an allosteric modulator is related to complex proteins characterized by a quaternary structure.
In the case of haemoglobin, there are 2 important allosteric inhibitors (they prevent the biological function:
the binding with O):
a. proton ions (in peripherical tissue),
b. Bisphosphoglyceric acid. The BPG has the correct size in order to enter inside the central cavity (of
the T state); moreover, it contains a phosphate group (negatively charged) useful in order to
perform weak interaction with AAs+ present on the surface of the cavity or to perform H bondings.
When the haemoglobin in T state binds the BPG, any movements of the polypeptide chains are
prevented, so this prevents the binding with O as well: even if there is oxygen, its presence isn’t
sufficient to bind to heme group because of the presence of BPG.
This kind of molecule cannot enter in the cavity of R state, which is too small.
In our body, BPG is used in order to favour the release of O (in peripherical circulation) and it is
normally injected in human body after a transfusion in order to force the new haemoglobin to
release oxygen.
Foetal haemoglobin
Foetal haemoglobin is similar to the adult haemoglobin except for 1 AA presents in the beta (which become
gamma) polypeptide chain: 1 histidine (+) is replaced with 1 serine (uncharged).
The histidine replaced is one of the central cavity involved in the interaction with BPG (which favour the
release of oxygen); the direct consequence is a major affinity for oxygen then mother’s haemoglobin. This
makes mother’s haemoglobin to release oxygen and foetal’s one to binds it: in general, the favour transport
of oxygen from mother’s blood to baby’s is favoured.
Bohr effect The Bohr effect explains how haemoglobin saturates in lungs and release oxygen in peripherical tissue.
49
The haemoglobin affinity depends on the concentration of 𝐶𝑂2. The Bohr effect says that a decrease of pH
(or increase in 𝐶𝑂2) reduces the haemoglobin affinity for oxygen; this is the condition of peripherical
tissues. On the contrary, the increase of pH (decrease in 𝐶𝑂2) decreases the affinity for oxygen; this is the
condition of lungs.
In peripherical tissue, cell consume oxygen and produce 𝐶𝑂2 and proton ions. As 𝐶𝑂2 is in the gas
phase, it has to be eliminated: this is achieved by the carbonic anhydrase, which turns the 𝐶𝑂2 to
liquid reducing it. This reaction forms a water molecule and a weak acid: the carbonyl acid; as every
acid, it dissociated into proton ions and its conjugated base. This increases the number of proton
ions and decrease the pH.
The haemoglobin present in the erythrocytes prevent any change in pH by binding the proton ions
in their central cavity. These are define as allosteric inhibitor for haemoglobin and they perform the
release of oxygen.
Erythrocytes move up to the lungs and experiment a change in the external environment: in lungs
there is an high oxygen concentration and a basic pH. For this reason haemoglobin tries to
neutralized the pH by releasing proton ions. This favour the protonation of carbonate in order to
form carbonic acid which is transform in 𝐶𝑂2 and water: the 𝐶𝑂2 of pheripherical tissue is
definitely eliminate by the body.
The release of proton ions from haemoglobin favours the interaction of heomglobin and oxygen:
because of the high concentration of oxygen in lungs, the interaction of thid with one chain is
favoured: the saturation of haemoglobin is performed is a short time.
With an high concentration of 𝐶𝑂2 (low pH, peripherical tissues) the oxygen binding curve shift towards
left: haemoglobin has a lower affinity for oxygen and releases it. When the 𝐶𝑂2 concentration decreases
(high pH, lungs) the oxygen binding curve shifts towars right: the affinity of haemoglobin for oxygen
increases.
HAEMOGLOBIN VARIANTS.
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There are a lot of possible mutation in haemoglobin AAs, but most of them are silent: 5% of world
population is affected by Hb mutation without any effect. This is because the import of the mutation
depends on the similarity of the two AAs involved and from their position: all mutations in a specific site of
binding cause a pathology.
Sickle-cell anaemia (SCA) is an hereditary disease, due to a mutation in the binding site. Visually, the
consequence is in the shape of erythrocytes: they aren’t spherical, but acquire a sickle shape.
In the aberrant haemoglobin 1 glutamic acid (-) in replaced by a valine (polar). This destabilizes all the
quaternary structure, because the glutamic acid should be involved in the weak interaction between
polypeptide chains, therefore so the interaction is reduced.
Moreover, the aberrant haemoglobin interact with others in order to form strands inside the erythrocyte,
which cause the typical shape. The pathological consequence is that such aberrant erythrocytes are not
able to move in vessels correctly and this get the blood flow to stop (ischemia).
This disease is dangerous if both haemoglobin genes are mutated.
Nevertheless, the possibility to have the mutation in half genome has a positive effect in population in
Africa, India, Asia and wherever malaria disease is spread. Indeed, malaria parasite reproduce itself inside
the erythrocyte and the different shape of pathological erythrocytes prevent its reproduction.
52
Membrane proteins: cell membrane
The cell membrane’s main function is to protect the cell
from external environment, indeed it surrounds the
cytoplasm of living cell, physically separating the
intracellular components from the extracellular
environment.
It is also involved in different cellular processes such as cell
adhesion, ion conductivity, cell signalling and attachment
surface for extracellular and intracellular structures.
It’s selectively permeable to ions and organic molecules
It is formed by phospholipids bilayer with embedded
proteins.
Phospholipid bilayer According to the fluid mosaic model, biological membranes
can be considered as a two-dimensional liquid in which lipid
and protein molecules diffuse; the polar lipid bilayers is
composed by specific biomolecules, called phospholipids (PLs).
PLs are the main components
of the cellular membrane and
have e peculiar structure. The
hydrophilic head contains the
negatively charged phosphate
group and glycerol and the
hydrophobic tails contains two
long fatty acid hydrocarbon
chains (repelled by water and
forces to aggregate).
In water, phospholipids may aggregate in different ways:
a. Liposomes are spherical vesicle characterized by a lamellar lipid bilayer (used for drug delivery);
b. Micelles are formed by phospholipids with short acyl chains which form a spherical pack shape in
which hydrophilic heads are outside and apolar chains inside;
c. Bilayer sheet are an arrangement into a quite fragile and thin two-layered sheet (tails pointing
toward the centre, which excludes water and molecules like sugar or salts)
The membrane can be in a liquid phase, where random walk exchange allows lipid to diffuse, or in a gel
phase, where lipid are locked in place. Increasing temperature is possible to pass from a del phase into a
liquid one.
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Dynamism of the membrane
Cellular membrane is characterized by a dynamism inside the proper structure due to the liquid phase of
the bilayer: this favour the movement of the whole membrane proteins. Therefore, the cellular membrane
can be defined as a fluid mosaic: it is fluid as there is free movement of components and a mosaic because
it’s composed by different biomolecules.
Membrane proteins There are different kind of membrane proteins: some are able to cross the entire cellular membrane,
others are present only on the surface (internal or external).
The major function of membrane proteins is to control the transport of metabolite; the selectivity
transport, important feature of the membrane, is conferred by membrane proteins: they can control the
movement of substrates inside or outside it.
Membrane protein could be divided into 2 class:
Extrinsic or non-integral: proteins which are able to interact with the phospholipids heads and
therefore are present on the external or internal surface.
They are quite well studied thanks to specific buffer able to solubilize them (mild aqueous solvent).
The possibility to solubilize in aqueous buffer is due to the high percentage of hydrophilic domain.
Intrinsic or integral (IMP): proteins which are able to strongly interact with phospholipids and can
cross the whole bilayer or bind to the tails.
As they can bind to tails, this means that they are characterized by hydrophobic AAs which are not
compatible with the previous buffer; for this reason, is difficult to solubilize this kind of membrane
proteins. A organic (polar) solvent is necessary in order to destabilize the binding between IMPs
and cellular membrane. The experimental problem is that it isn’t possible to use strong organic
solvents, so the used ones have a low extraction power and their efficiency is low. In conclusion,
this proteins are difficult to characterized.
These can divided into:
o Properly integral proteins are able to cross the entire membrane: they have one
hydrophobic domain (which interacts with the hydrophobic domain of phospholipid) and 2
hydrophilic domains;
These are divided into 3 main families:
1. Single-pass membrane proteins can cross once the bilayer through a-
helix motif:
i. Type 1 is characterized by a C terminal present in the cytosol;
ii. Type 2 is characterized by a N terminal present in the
cytosol.
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2. Multi-pass membrane proteins can cross more than once the bilayer:
i. Type 3 has one polypeptide chain: different domains are
connected by loops;
ii. type 4 has more than one polypeptide chains: all
transmembrane domains are present in different chains able
to interact each other by a specific tertiary structure which
perform the final 3D structure.
3. Very complex proteins characterized by an high numbered of
alpha helices or beta sheet motifs can cross the membrane and
create a channel (beta-barrel).
o Lipid anchored proteins are hydrophobically insert into the membrane
and anchor to it by covalently linking to lipids; it consists in only one
extra-cellular domain. It is associated to the membrane from one side
but do not span the lipid bilayer completely.
These are divided into 4 main class:
1. Specific amphiphilic alpha helices able to interact both with phospholipid heads
and tails;
2. Hydrophobic loops which connects different alpha helices and bind to tails;
3. Proteins able to covalent bind one phospholipid and favour lipidation reaction;
4. proteins characterized by hydrophilic AAs able to interact with the hydrophilic head
through ionic and Van der Waals interactions.
55
Transport across membranes The cellular membrane is semipermeable membrane and could favour the transport of only specific
molecules without the presence of proteins.
The spontaneous movement of substrate is
controlled by a chemical (for non-charged
molecules) or charge (for ions) gradient and
occurs through diffusion.
A spontaneous process is the movement of
one uncharged molecule from a high
concentrated compartment to a less
concentrated one in order to reach an equilibrium. Equally, the spontaneous movement could occurs for
ions: it happens form an high ions concentrated environment to a less concentrated one in order to reach
an equilibrium of charges.
In all spontaneous movement, there is a negative Free Gibbs Fnergy as the transport follow one of a
gradient in order to reach an equilibrium.
There are different categories of transport for uncharged molecules:
Free transport occurs when a substrate is able to cross the cellular membrane and can diffuse
across it. This isn’t the trivial method as just few molecule can perform it (O and CO2).
Mediated transport is controlled by specific membrane proteins called carrier (or transporter).
This could be divided into:
o Passive transport is a movement that doesn’t requires energy and follows the gradient
(negative Free Gibbs Energy). It occurs through
a. Transmembrane carrier, which are fixed and cross the whole membrane;
b. Carrier ionophores, which can mov inside the cellular membrane.
o Active transport doesn’t follow the gradient and requires energy to move substrate from a
compartment with a low concentration to a compartment with high concentration (against
the gradient). It is characterized by a positive Free Gibbs Energy.
It can be
a. Primary active carries, where molecules use the energy released by specific
molecule (ATP);
b. Secondary active carriers, where the transporter couple the movement of a ions
down its electrochemical gradient to the uphill movement of another molecule or
ions against a concentration/electrochemical gradient.
The most used method is the mediate transport, even if it requires the presence of carriers; this feature is
clear considering the Free Gibbs Energy required for both phenomenon.
In the cytoplasmic compartment the substrate is characterized by one hydratation layer able to stabilize the
molecule; the number of water molecule able to interact with substrate’s surface depends on the number
of hydrophilic domain.
The diffusion requires the elimination of such hydratation layer, but this operation destabilize the
substrate; therefore, the first step of transport is characterized by an high amount of energy.
56
The substrate deprived of the hydratation layer could now
cross the membrane. During the molecule’s cross, the energy
required is constant as it depends on its degree of
hydrophobicity: while hydrophobic domains are compatible
with the internal bilayer, hydrophilic ones require more
energy.
Once inside the cell, the substrate reform its hydratation layer
and this requires energy.
The movement of the same substrate by using one membrane
carrier is lower. Indeed the spectra is characterized by the same
peak of energy as all step are the same, but the amount of
energy is completely different and it experiment a reduction of
Free Gibbs Energy.
Transporters There are different types of mediated passive / active
transporter:
a. Uniporters are transmembrane proteins able to transport
only one molecule of substrate.
b. If a transmembrane protein can transport 2 or more molecules it could be defined as
a. An antiporter if it’s able to move two or more different molecules or ions across a
phospholipid membrane in opposite direction
b. A symporter if it’s able to move two or more different molecules or ions across a
phospholipid membrane in the same direction.
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Mediate passive transport: CARRIER IONOPHORES One example of mediate free transport in cells is called valinomycin and it is formed
by valine AAs; it is a potassium-specific transporter and facilitates the movement of
potassium ions through lipid membranes in according to the electrochemical potential
gradient.
The position of different AAs allows to understand how it move inside the membrane
and how it binds the potassium ions. On the external surface there are the hydrophobic residues of valine:
this confers the ionophores the possibility to move.
In the central cavity it can bind K ions thanks to the
carbonyl oxygen atom, characterized by one
negative pole able to interact with potassium
through Van der Walls interaction.
The specificity is due to the number of AAs as they
confer the specific dimension to the central cavity,
specific only for K ions.
Mediate passive transport: AQUAPORINS
Aquaporins are Imp type III (one polypeptide chain) integral
membrane proteins. They are characterized by 6
transmembrane domains with an alpha helix secondary
structure (which allows to cross the membrane as hydrophobic)
and 5 interhelical loop regions. Aquaporins can interact each
other in order to form a central channel which can selectively
conduct water molecules (1 per pore) in and out of the cell,
preventing the passage of ions and other solutes.
In the primary structure there a specific AAs triplet: asparagine-
proline-alanine. These are sequentially far each other as are
present in the first, second and last loop, but they overlap during the folding into the narrow pore creating
an internal channel.
The tertiary structure sees the alpha helices interacting with the entire
cellular membrane as they are hydrophobic and a central core with the
triplet.
Considering the 3D conformation, there are 2 major domains:
• 2 conical domains, one in the external and one int the internal
membrane surface. Such domain are selectivity and stabilize the water
forming H bindings with water molecules. Therefore they are characterize
by specific AAs (aromatic and arginine) which can perform H bindings
reducing the Free Gibbs Energy. They act as selectivity filters.
• 1 narrow domain, characterized by the presence of 2 NPA motif
able to interact with the water molecules. Its narrowest prevent the H
binding between different water molecule and select just 1 molecule.
1. The canal start with a conical pore, which consist in the first selectivity filter. The presence of
aromatic AAs and arginine allows the domain to stabilize the water molecule as it loses the
hydrogen bindings with the other 4 water molecule whom its usually bond; this allows to reduce
the instability created perturbing the still situation introducing the water molecule in the filter. The
58
final effect is that the water molecule is approaching the membrane protein reducing its
hydratation layer, but preserving its stability.
2. As the water molecule enters in the canal, the number of H bindings with other water molecule is
reduced, while the number of H bindings with the filter increases.
3. In the narrow domain, the canal dimension in complementary to the molecule dimension:
aquaporin is specific for only water molecule. Here, all H bindings are disrupted and the molecule is
stabilized by new H bindings with the central pore motif.
4. Then, the water molecule arrives in the second selectivity filter, which favours the progressively
creation of new H bindings with other aromatic AAs and later with water molecules: the water
molecule ca recreate its hydratation layer in the cytoplasm.
Glucose transporter: GLUCOS TRANSPORTER 1 There are different type of glucose transport in order to guarantee the right amount of glucose to permit
metabolism; GLUT1 is a uniporter molecule that transports glucose across the plasma.
It is a type III integral protein with 12 hydrophobic segments (12 membrane-spanning alpha helices)
assembled to produce a transmembrane channel with hydrophilic residues able to hydrogen-bond with
glucose.
It exists in 2 stable conformation: T1 is characterized by the possibility to bind glucose from extracellular
environment, while T2 to release into the cytoplasm.
It’s a passive transport: such transporter is activated only when cell requires energy, that is when glucose
contraction inside is lower than outside. The transport occurs through a conformational change.
1. Glucose from blood plasma binds to stereospecific site in T1 (reduction of free energy required for
transport);
2. Conformational change from T1 to T2 (glucose transmembrane movement);
3. Release of glucose in the cytoplasm 4. Return to T1 conformation to start another transport cycle
The velocity-concentration curve of glucose could be compared to the mannitol one. This is transported in a
is completely different way: by free diffusion; anyway, for both molecules, transport inside the cell is
strictly related to their contraction outside.
59
The velocity of glucose transport is faster and tries to achieve a maximum velocity: the saturation of all
transporter for glucose; as cell has got enough glucose transporters to satisfy its need, the asymptotic value
wold never be reached.
Moreover there is a difference considering low substrate concentration: in mannitol, the velocity is low at
low concentration while in glucose is higher; this is because the metabolism is a continuous process and has
to be achieved in a short time.
Another feature is the possibility to control the transport by allosteric modulators; these is another reason
why cells prefers the mediated transport: it could be controlled in a short time considering the request of
the cell.
Mediated primary active transport: 𝑁𝑎+/ 𝐾+- ATPase The sodium potassium pump (or ATPase) can use the energy released by the hydrolysis of ATP in order to
transport different ions against their gradient. It is an antiport protein: it carries inside 2 K ions and outside
3 Na ions.
1. The pump, while binding ATP, binds 3 intracellular Na+ ions.
2. ATP is hydrolysed, leading to pump’s phosphorylation at aspartate residue and subsequent release
of ADP.
3. A conformational change in the pump exposes the Na+ ions to the outside (phosphorylated pump
has a low affinity for Na+ ions).
4. The pump binds 2 extracellular K+ ions, causing the loss of phosphoric group and a consequent
conformational change.
5. The dephosphorylated pump has a higher affinity for Na+ ions than K+ ions, so the two bound K+
are released. ATP binds and the process starts again.
The general reaction is:
The number of positive charges that are transported outside and inside is different: the function of ATPase
is to reduce the positive charge inside the cell; this allows the cell to maintain the physiological potential of
cellular membranes.
Moreover, the selective permeability of the cell's plasma membrane for ions permit to maintain the cellular
volume, which is mostly due the content of water inside the cell. If cell’s osmolarity (ions concentration) is
higher inside than outside, water flows inside cell (in order to forma proper hydratation layer) through
osmosis causing cell’s lysis.
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Enzymes Enzymes are little globular proteins, characterized by a low MW and are involved in the catalysis of specific
reactions. The catalysis reduces the time of the reaction and increases its rate (speed).
Different reaction are catalyse by different enzymes as they can recognize
one specific substrate.
The possibility to recognise one specific enzyme is the first and limiting
step of the reaction. This includes a binding between the enzyme and the
substrate in order to form the complex ES. After this bond, the substrate is suddenly transformed into a
product and the enzyme is released as the new compound is different from the specific substrate recognized
by the enzyme.
The specificity is due to the fact that the 3D
structure of the active site is complementary to
the one of the substrate.
The bind is due to the active interaction of the
chemical group of the substrate and those
present inside the active site. This latter’s are
moved in a precise conformation thanks to the
reshape of the enzyme; this provide a specific and precise interaction with substrate. The formation of
specific weak interactions require time, for this reason the formation of ES is a limiting step.
This above are the reason why enzymes could be defined as specific and selective biomolecules.
Enzymes can change a specific thermodynamic
parameter present in the reaction. These are the
variation of Free Gibbs Energy and activation
energy.
The Free Gibbs Energy – both initial and final
– it’s the same with or without the enzyme, as it’s
specific of the substrate. Therefore the difference
of Free Gibbs Energy is the same and this means
that the type of the reaction remains the same
(endergonic or exergonic) and maintains the
concentration of reaction compounds at
equilibrium.
The enzyme reduce the activation energy:
the potential energy required to change the substrate into a specific intermediate (transition state),
which is instable, but required for the production of the final product.
In conclusion, the difference between the uncatalyzed and the catalysed reaction is the value of the
activation energy: the catalysed substrate requires a lower amount of energy in order to become the TS. For
this reason, the reaction’s speed increases.
Catalyse reaction are preferred to the uncatalyzed one (especially the metabolic ones): the first reason is that
an higher speed is preferable, moreover they can be controlled through activator.
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Kinetic In general, enzymes are able to increase the reaction rate.
The saturation reaction rate is an hyperbolic curve which
describes their behaviour.
at low substrate concentration, there is a quite low
reaction rate,
up to a substrate concentration specific for each
enzyme, the curve starts to increase and almost achieve an
asymptotic value called maximus velocity.
Biologically,
when the substrate concentration is low, the
catalysis is not so efficient as only a low number of
enzymatic molecules will be involved in the process. For this
reason the reaction rate increases quickly with an direct
proportion with the substrate concentration: an higher
substrate concentration enlists free enzymes.
Up to the saturation of all enzymatic molecules, when all enzymatic molecules are involved in
the formation of the ES complex and it’s possible to move closer (but slower) to the maximum
velocity.
In physiological condition it isn’t possible to reach the asymptotic value as not all enzymatic
molecules form the ES complex (the first reaction has an equilibrium indeed); this is because it is
possible that some active complex ES aren’t stable and they dissociate.
The reason of the backward reaction is that not all the ES complex are stable and able to transform the
reagents into the products. Indeed, the substrate has to fit inside the active site and interact with specific
chemical group; if the substrate interacts with a different area, the interactions involve different chemical
groups and the ES isn’t stable.
There are some specific parameters:
𝑉𝑚𝑎𝑥 is the theoretically maximum velocity reachable by the enzyme;
Michaelis-Menten constant 𝐾𝑚 is referred to the affinity of the enzyme for the substrate: is the
substrate concentration required for an enzyme to reach one-half its maximum reaction rate.
This can be find is specific tables and it is a characteristic for each substrate.
Catalysis constant 𝑘𝑐𝑎𝑡 is the concetration of product formed in 1 actvie site in 1 second. It represent
the capacity of the enzyme in catalysing the reaction.
Specificity constant is the ratio between 𝑘𝑐𝑎𝑡 and 𝐾𝑚 and it’s able to correlate the affinity for the
enzyme for the substrate and the catalytic action. Tt is useful to compare different enzymes or the
same enzyme with different substrates
A catalytically perfect or kinetically perfect enzyme is an enzyme able to perform all stable ES
complex in order to transform all bound substrate into products. This means that this theoretical
enzyme are able to interact in a perfect way with the substrate: every collision enzyme - substrate
will form ES complex and the product formation depends only by diffusion rate.
These enzymes have a very high specificity constant.
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Cofactors Another feature of the enzymes is the possibility for them to function in couple with specific small molecules
called cofactors; this could be ions or other molecules able to favour the enzymatic catalysis; without the
cofactors enzyme doesn’t work.
NAD is a very diffused cofactor present in redox reactions
as it is able to bind or release electrons. All redox reactions
use cofactors as acceptors/releasers of electron; indeed a
redox reaction occurs always coupling two reactions: one
reagent releases electrons (reduction reaction) and
another acquires them (oxidation reaction).
The reason why all physiological redox reactions use
cofactor is that electrons can’t be left free in the cell, apart
from specific situations.
From a structural point of view, the NAD is a dinucleotide:
it is composed by 2 molecules of ribose and 2 nitrogenous
bases (adenine and nicotillamide). This 2 domain are
linked each other through 2 phosphate groups.
NAD can be simple or phosphorylated (NADP).
The redox of NAD is coupled with another reaction present in the reaction cycle. In the reduction of NAD, N
atom has a positive charge and can acquire 2 proton ions and 2 electron ions released by the oxidation of
another biomolecule losing its positive charge. NAD could also be oxidize: it release the proton and the
electron ions which can be acquired by biomolecules.
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The rate law The reaction law is a relation able to link the reaction rate with the concentration of the substrate.
Considering one general reaction with 2 different substrates able to be transformed into one product, the
rate low is:
𝑎𝐴 + 𝑏𝐵 → 𝑐𝐶
𝑟 = 𝑘[𝐴]𝑥[𝐵]𝑦
Where [𝐴] and [𝐵] are the molarity of the substrates, 𝑥 and 𝑦 are experimentally determined (they aren’t
the stoichiometric coefficients) and K is the rate coefficient or rate constant, dependent on experimental
conditions (T, pH…).
Reaction can be divided into 3 categories according to their rate law:
a. The 0 order reactions are characterized by a rate law independent from the substrate concentration,
but only from experimental condition:
𝑟 = 𝑘
Fixed the experimental conditions, the rate is fixed as well.
b. The first order reactions have a rate law which depends on the concentration of 1 substrate and on
𝑘. Other reactants can be present, but each will be zero-order. It is possible to change the reaction
rate changing the substrate concentration.
𝑟 = −𝑑[𝐴]
𝑑𝑡= 𝑘[𝐴]
This is also called unimolecular reaction.
c. The second order reaction have a rate law which depends on the concentration of 2 different
substrates – or on only the concentration of one substrates elevated at the second order – and on 𝑘.
Little changes in substrates’ concentration could favour the suddenly increase of rate.
𝑟 = −𝑑[𝐴]
𝑑𝑡= 𝑘[𝐴]2
𝑟 = −𝑑[𝐴]
𝑑𝑡= −
𝑑[𝐵]
𝑑𝑡= 𝑘[𝐴][𝐵]
Single-substrate reactions: Michaelis-Menten kinects Even if a single substrate is involved, the presence of ES intermediates favours a ping–pong mechanism (as
not all ES complex are stable: the bind is not functional and the enzyme releases the substrate), typical of
multi-substrate reactions. This mechanism isn’t present in perfect enzymes.
In order to reduce this ping pong mechanism, which can
reduce the reaction rate, a specific concentration of substrate
and enzymes is used.
For this reason, all
studied reaction are
carried out at high substrate concertation and low enzyme
concentration in order to favour the formation of stable ES complex.
Indeed, in this condition, even if an ES complex isn’t stable and the
enzymes release the substrate, another substrate will suddenly be
bind. In this condition the ES complexes’ concentration is constant and
can be considered a steady state. This is also true in physiological
condition
This increases the rate and the concentration of final product.
64
Michaelis-Menten constant (Km) The Michealis constant is able to consider the first limiting step and the transformation of substrate into a
product.
The concentration of total enzyme is a sum between the free enzyme and the one in ES complexes:
[𝐸]𝑇 = [𝐸] + [𝐸𝑆]
without considering the final free enzyme as the substrate concentration is so high that the free final E is
immediately bind to substrate.
𝐾𝑀 =𝐾−1 + 𝐾2
𝐾1
𝑉𝑚𝑎𝑥 - Michealis-Menten equation
𝑣 =𝑉𝑚𝑎𝑥[𝑆]
𝐾𝑀 + [𝑆]
This means that when 𝐾𝑀 = [𝑆] , 𝑣 =𝑉𝑚𝑎𝑥
2
Catalysis constant 𝐾𝑐𝑎𝑡 The catalysis constant is defined considering a concentration of S higher thank 𝐾𝑚.
𝑣 =𝑉[𝑆]
𝐾𝑀 + [𝑆]
Considering the saturation curve, in this condition 𝑉 = 𝑉𝑚𝑎𝑥 as 𝐾𝑚 is negligible; therefore the reaction
becomes independent on [S] and with a zero-order kinetics. The reaction rate asymptotically approaches
maximum rate.
𝐾𝑐𝑎𝑡 =𝑉𝑚𝑎𝑥
[𝐸]𝑇
The 𝐾𝑐𝑎𝑡 depends on 𝑉𝑚𝑎𝑥 and the concentration of enxymes. For this reason is important to fix 𝑉𝑚𝑎𝑠 in
order to fix the type of enzyme.
𝐾𝑐𝑎𝑡 is referred to the second kinetic constant, able to control the second part of the reaction (substrate
into products) (𝑘2).
Specificity constant the efficiency of an enzyme can be expressed in terms of specificity constant, which reflects both affinity and
catalytic ability. It compares the affinity of one enzyme for a specific substrate and its ability to transform the
substrate into products.
It’s measured in experimental condition when [S] is lower than the 𝐾𝑚. The reaction rate depends by both
concentration of E and S, so the reaction rate could be defined as a second order.
65
Enzyme activity control
The control of the enzyme activity is essential for the steady state of all organisms therefore enzymatic
activities can be either up-regulated or down-regulated; this controls the modulation of cellular pathways to
prevent either uncontrolled growth or catabolism.
Enzyme’s catalysis is regulated controlling:
1. Enzyme quantity: long term control involving the possibility to change the enzyme’s grade of
synthesis. The increase or decrease of genetic transcription requires time, but has very long term
effects. It has to do with gene regulation.
Normally, such genetical modulation is not present during the normal life of a cell, but it’s typical of
pathological conditions.
2. Enzyme activity: short term control important in order to increase or reduce the enzymatic function
without changing the quantity of enzyme.
The enzymatic synthesis and the condition are the same, but specific molecules can directly interact
with enzymes in order to increase or reduce their activity.
Short term modulation
There are 3 main categories of short term control:
1. Enzyme inhibition occurs through inhibitors or activators, which are molecules able to interact with
a specific site in the enzyme, that could be the active site.
2. Allosteric modulation involves other specific molecules which can interact with the enzyme binding
in specific allosteric site, different and far from the active site.
3. Covalent modulation controls the enzymatic activity by changing its final 3D conformation. It requires
the possibility to transfer 1+ AAs by a phosphorylation or 1 specific sugar molecule; the change of
the final composition of the enzyme involve the change of the 3D conformation which changes its
biological activity.
1. Enzyme inhibition Inhibitors are able to directly bind to the enzyme in order to prevent the transformation of the substrate into
the products.
Inhibitors and activators could be endogenous, characterised by weak interactions or exogenous. The
endogenous inhibitors’ function is reversable and the enzyme doesn’t permanently lose its function; for this
reason, this kind of modulation requires the possibility to disrupt the interaction to release the enzyme which
can continue its catalytic function.
On the contrary, exogenous inhibitors (drugs) are irreversible as their binds are strong (e.g. covalent); their
function is to reduce and stop the enzymatic activity permanently.
Endogenous inhibitors are classified as
a. competitive,
b. uncompetitive or
c. non-competitive inhibitors,
as they interact in different moment of the enzymatic catalysis.
In this case, the inhibitor and the enzyme act independently of each other.
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a. Competitive inhibitors could act in 2 different ways:
They could be molecules which can
compete with the substrate in binding the
enzyme; the majority of them are able to
interact directly with the active site
through weak interactions. For this
reason, their structure is very similar to
the substrate’s one.
They interact with the free enzyme: the complex enzyme-inhibitor prevents any next
reaction.
On the other hand, other molecules could
bind to a site close to the active site hiding
the active one and preventing its
interaction with the substrate.
In this case the inhibitor has a quite
different 3D structure in comparison with
the substrate.
In this case the Michaelis-Menten constant 𝐾𝑚 increases:
due to the interference of the inhibitor, the substrate
concentration needed in order to reach half of 𝑉𝑚𝑎𝑥 is
major.
On the contrary, the maximum velocity 𝑉𝑚𝑎𝑥 remains the
same because when the substrate is able to bind the
enzyme (high concentration of substrate), it acts in the
same way as without inhibitors.
Example. The alcohol dehydrogenase is the enzyme which convert ethanol into acetone and then
acetic acid, which is physiologically released by urine and is non toxic.
A competitors for the ethanol is the methanol, which has an higher affinity for alcohol
dehydrogenase; such interaction produces dangerous molecule formaldehyde.
The treatment for methanol poisoning is the administering of ethanol, which is equally an inhibitor
for methanol in the reaction through alcohol dehydrogenase.
67
b. Uncompetitive inhibitors don’t compete with the
substrate, but interact with the complex enzyme-
substrate; the formation of such super-complex prevents
the formation of products.
Both the Michaelis-Menten constant 𝐾𝑚 and the 𝑉𝑚𝑎𝑥
decrease. The first decrease as 𝐾2 decreases, the latter
because the possibility to transform substrate into
products decreases.
c. Non-competitive inhibitors are able to bind both the free enzyme and the complex ES.
The Michaelis-Menten constant 𝐾𝑚 isn’t affected even if 𝐾2
decreases; this is because in this condition there is the balance
between two different actions: when the non-competitive
inhibitors act as a competitive, the 𝐾𝑚 increases, when it act as
uncompetitive, the 𝐾𝑚 decreases.
The 𝑉𝑚𝑎𝑥 decreases because the concentration of substrate
which can be transformed into product deceases.
2. Allosteric modulation Allosteric modulation is possible in complex enzyme formed by
different subunits (quaternary structure) as these are characterized by
2 stable state: T-state (inactive) and R-state (active). These 2 different
states have different reaction rate.
It is based on allosteric modulators: specific molecules able to interact
in the allosteric site, a site far from the active one ( inhibitors).
The interactions are weak and are able to change the 3D conformation of the entire enzyme which changes
the quaternary structure.
Example. The ATCase is an high regulated enzyme able to combine 2 different substrates in order to obtain
the first product of the pyrimidine biosynthesis. This metabolic pathways is characterized by the
condensation of aspartate and carbamylphosphate to N-
carbamyl-Laspartate and inorganic phosphate.
ATCase controls the rate of pyrimidine biosynthesis by
altering its catalytic velocity in response to cellular levels
of both pyrimidines and purines.
The end-product of the pyrimidine pathway, CTP, induces
a decrease in catalytic velocity (retroactive inhibition),
whereas ATP, the end-product of the parallel purine
pathway, exerts the opposite effect, stimulating the
catalytic activity.
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The ATCase has 12 domains:
Catalytic subunits are characterised by active sites: they link
and transform the 2 substrates. They are composed by 2
trimers: C1, C2 and C3 and C4, C5 and C6.
Regulatory subunits are involved in the regulation of the
enzymatic activity and in the formation of the stable T state.
The contains binding site for ions (Zn) and the allosteric
domains.
The relaxed R state has an high affinity and high activity action: the
catalytic subunits bind the 2 substrate in order to transform them in
1 product.
On the contrary, the tight T state is completely different: a shift of subunits prevents the binding of substrate
in the active sites.
ATCase swells in size during the allosteric
transition and the catalytic subunits condense
during this process: catalytic chains come closer
together (better contact with substrates)
When the concentration of the final product is high, the metabolic pathway could be reduced: the binding of
CTP to the regulatory subunits results in an equilibrium shift towards the T state.
On the contrary, it is possible to have shift the equilibrium through the R state, thanks to the binding of ATP.
2. Covalent modulation Covalent modulations is a quite strong way to control the enzyme changing the enzyme’s 3D conformation
by covalently binding a chemical groups. Its results could last for a ling time has the releasing of the chemical
compound requires energy to disrupt the covalent binds.
All this modifications are controlled by an enzyme.
The most common covalent modification is the
phosphorylation; but also glycosylation and methylation are
used.
Enzymes that control metabolic pathways can be subject to
reversible regulation by phosphorylation of specific sites.
Phosphorylation usually results in a functional change of the
target protein (substrate) by changing enzyme activity or
cellular location.
Normally, the phosphorylation is performed by kinase, while phosphatase have the opposite function.
Kinases are enzymes able to modify other proteins by chemically adding phosphate groups to them from
ATP(phosphorylation).
69
Usually, the source od the phosphate group is the ATP, thus it requires energy.
Example. Glycogen phosphorylase is involved in both allosteric and covalent modulation (reversible
phosphorylation) in the glycogenolysis.
Glycogen is a glucose polymer and represent an energy storage; when in need for energy, glucose is releases
1 molecule per time by glycogenolysis.
Glycogen phosphorylase it’s a very complex and large enzyme; while the enzyme can exist as an inactive
monomer or tetramer, it is biologically active as a dimer of two identical subunits (phosphorylase a
(phosphorylated) + phosphorylase b (unphosphorylated).
Both subunits contain:
The catalytic sites, which are relatively hidden; this lack of easy access of the catalytic site to the
surface makes the protein activity highly susceptible to regulation.
Glycogen binding sites (covalent interactions), close to the catalytic site; these are necessary as
glycogen has an high sterical hindrance
Allosteric sites, far from the active sites. Binding of AMP at this site, corresponding in a change from
the T state of the enzyme to the R state, results in small changes in tertiary structure at the subunit
interface leading to large changes in quaternary structure.
Ser14, as serine are the AAs involved in the reversible phosphorylation.
The phosphorylation is the main up-regulator of the pathway and is able to change the conformation of the
enzyme from T into R state.
Anyway, not all enzyme molecules can be phosphorylated and remains the phosphorylase b which is inactive
(T state). Nevertheless, if the cell requires more energy the activity of phosphorylation of these would be
favoured by specific allosteric activators (AMP, ADP).
On the other hand, when cell is saturated with energy, the activation of phosphatase remove the phosphate
group from the enzyme: even if this unphosphorylated almost all enzyme molecules, those still in the R state
are forced to change conformation through allosteric control.
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Serine proteases
Serine proteases are enzymes divided into 4 main categories and each categories could be divided into many
families. Proteases are enzyme able to perform a proteolysis, which is the destruction of proteins through
the digest of peptide bonds. Cell requires the digestion of proteins in order to perform the catabolism
(planned destruction of biomolecules), but sometimes proteolysis also occurs during the activation of
proteins and enzyme, as it could be involved in their modulation.
Serine proteases are characterized by a large number of types because they are involved in many different
biological functions (digestion, immune response, blood coagulation and reproduction).
They are all characterized by the presence of one serine AA inside the catalytic
site.
Moreover, they have the same quite complex 3D conformation as they share the
biological function: 2 beta-barrels domain, formed by different beta-sheet
domain, are able to interact each other in order to form a catalytic site in their
middle.
The difference between the four categories lies in the ability to interact with different AAs: what changes is
the dimension of the binding site, which is very close to the active site.
i. Chymotrypsin-like proteases are characterised by the possibility to interact with large
(aromatic) AAs because in their binding site there are very small AAs (serine) an hydrophobic
AAs.
ii. Trypsin-like proteases are able to bind positive charged AAs because they contain 1
aspartic acid (hydrophilic, negative).
iii. Elastase-like are able to bind very small AAs (alanine, glycine) because they contain
quiet small AAs characterozed by a not so high sterical hindrance
iv. Subtilisin-like proteases are able to bind quiet large and aromatic AAs. Even if it’s typical in
prokaryotes, it has a catalytic mechanism similar to the chymotrypsin. This is because they derive
form the same ancestor precursor: although the have had different
evolutionary process, they have conserved the AAs present in the
active site, even changing their primary structure. This process is
called convergent evolution.
Even species very far one from each other will contain proteins
which might be different from a general point of view, but
characterized by the same AAs in the catalytic site. Moreover, even
if the AAs sequence is very different, the different AAs are able to
fold in a similar 3D conformation.
71
Catalytic mechanism of serine proteases Catalytic mechanism happens in the catalytic triad located in the active site of the enzyme; it is formed by 3
AAs: histidine, serine and aspartic acid. Each of them is important in a step of the catalytic mechanism, but
the serine has the principal role in the hydrolysis. Considering their position in the primary structure, they
are normally far form each other, while after the fold the are all present in the active site.
This particular geometry characterizes a ping-pong mechanism, which involve a movement between
substrate and product inside and outside.
The first step is the binding of the substrate after the hydrolysis of the peptide bond; after the release of the
first product, a water molecule enter inside and cause the release of the final product.
In the catalytic site there is serine, histidine (secondary role: creates instability) and one aspartic acid which
is involved in the hydrogen bonding with the hydrogen bonding with the N atom of histidine which is not
directly involved in the hydrolysis (up) and increase the reactivity of the other N, involved in the catalytic
function.
i. The substrate is previously
bound to the binding site, so the
peptide bond to be cleaned is
present inside the catalytic site.
The first interaction is a bound
between the carbon atom of
peptide bond and the oxygen of
histidine.
Moreover, this release his H
because the N of histidine
perform one H bond which is
lately transformed in a covalent
bond.
ii. When the H of serine is
captured by the covalent bound
with histidine, another covalent
bond can occur between the O of
the serine and the C atom of the
peptide; this allows the digestion.
iii. This second bond causes an
high instability which digest the peptide bond: the first product to be release in the N terminal as the
C terminal is covalently bound to serine.
iv. After this, 1 water molecule enter inside the catalytic site and perform 1 hydrogen bonding with
the H of histidine, which is useful in order to create an interaction with the C terminus peptide.
v. Then the O of water perform a covalent bond with the C of the peptide leaving its H to the
histidine; this forms a second instable intermediate which broke the previous covalent bond with the
O if serine.
vi. The second product (C terminable) is now released and the H bound to the histidine, which was
previously bound to water, is transferred to the serine in order to create another active site.
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While the hydrolysis action is performed by
the triad, other AAs are useful in order to
stabilize the catalytic mechanism performing
specific weak interactions with substrate. For
example, glycine can stabilize the substrate
inside the catalytic site, performing a an
additional hydrogen bonding with the
carbonyl group of substrate before the
hydrolysis.
Regulation of serine proteases activity Serine proteases are involved in different processes of regulation and therefore they are strictly controlled
as their activity could become dangerous when excessive. There are 2 kind of serine proteases regulation:
zymogens activation or enzymatic inhibitors.
Zymogens activation The first possibility to control serine protease is the possibility to synthetize inactive precursor called
zymogen. They are large molecules that can be transformed into the smaller activated enzymes by cutting;
the active site for catalysis is different between zymogens (distorted) and the activated enzymes and
consecutively substrate can’t bind effectively and proteolysis doesn’t occur.
Activation consists in the change of zymogen’s conformation and structure which implies the open of the
active site.
There are several protective measures taken by the organism to prevent self-digestion, one of this is that
zymogens are stored in zymogen granules, capsules that have resistant walls to proteolysis. In this way, the
proteolysis in cell occurs only when required.
The most important activation is the one of trypsin, which
occurs by enteropeptidases, secreted in the duodenal
mucosa. These are able to recognise the peptide bond
present in trypsinogen involved in the formation of the
additional sequence of AAs (lysine and isoleucine) and
release the active trypsin and the additional sequence of AAs
(which increase the activity of enteropeptidase). Trypsin
could start to digest specific proteins and favour the auto-
activation: it could recognize its zymogen and digest the
specific peptide bond in order to release active trypsin.
73
Trypsin is a quite interesting enzyme as widely diffuse in body
and is also able to activate the other serine protease present in
our body: they can recognize chymotrypsinogen and proelastase
and digest the specific peptide bond in order to release
chymotrypsin and elastase.
Chymotrypsinogen is characterized by just 4 additional AAs
compared to the active protease; trypsin recognizes such
sequence, hydrolyses 4 peptide bonds in order to release 1
couple of dipeptide and obtain the chymotrypsin active primary
sequence. The feature of chymotrypsin is that there isn’t one
homogeneous primary structure, but this’s due to 3 different
sequence of AAs, all bound through disulphide bridges.
Enzymatic inhibition The second way to control serine
proteases is the possibility to
synthesize specific inhibitors, which
stop their function interacting with
it. The endogenous inhibitors are
called serpsin and are able to
covalently bind the enzyme only
after the binding with substrate
(uncompetitive inhibitors).
Endogenous inhibitors are
reversable: the enzyme doesn’t
completely lose its function;
otherwise, un-reversable inhibitors
are called PMSF.
THROMBIN Thrombin is a human serine protease able to
convert soluble fibrinogen into insoluble
strands of fibrin and to catalyse many other
coagulation-related reactions; its zymogen is
prothrombin (coagulation factor II), cleaved
to form thrombin in the coagulation cascade.
The coagulation cascade is performed after an endogenous or exogenous injuring of vessels, which is the
disruption of endothelial tissue and causes the loss of blood.
74
Any injury releases the tissue factor which start the coagulation
process; this is an huge number of connected reaction with
activate the thrombin (secondary haemostasis). When it start,
there is the contemporary interaction of platelets, which forms
a plug at the site of injury (primary haemostasis). This plug is
not so stable but it stabilize due to the action of fibrin, which
perform a sort of matrix.
The tissue factor is able to activate a large number of enzymes
called factors important if the final activation of thrombin. The
thrombin is usually stored in zymogen vesicle in one inactive
zymogen called prothrombin, characterized by an high number
of AAs, able to prevent its action.
After the activation of thrombin, it could activate the fibrin,
which is stored in inactive precursor called fibrinogen. The
activation of fibrinogen into fibrin monomer starts the activation
of other fibrin monomers, forming the fibrin fibres which interact
each other form a crosslinked polymer.
The mechanism is the following:
i. The thrombin is able to recognise fibrinogen, formed by an hexamer, which contains 2 sets of 3 pairs
of non-identical polypeptide chains (alpha, beta and gamma chains) linked to each other by
disulphide bonds. Thrombin recognise additional AAs sequence in alpha and beta chains and digest
such specific peptide molecules. The elimination of part of alpha and beta chains convert the
fibrinogen into active fibrin monomers.
ii. Different fibrin monomers are able to interact each other in order to form protofibrin, a linear
sequence of different fibrin monomer.
iii. Different protofibrins interact each other in order to obtain fibres,
iv. which interact each other are able to perform cross link covalent bond between different protfibrin;
these are useful in order to produce branches.
In conclusion, the main function of thrombin is the activation of fibrin and the stabilisation of platelets.
Moreover, thrombin can also increase the aggregation of platelets as they contains specific receptors on their
surface and can increase their interaction in order to increase the dimension of the plug present in the
damage tissue.
In addition, another important function starts at the end of the coagulation process: the inhibition of
coagulation through the activation of anti-inflammatory factors. Thrombin binds to trombomodulin and the
complex activates the protein C which start the anti-inflammatory process which is useful in order to inhibit
the coagulation. This function controls the coagulation process as well.
Cell can also modulate the function of thrombin by the activation of specific inhibitor: antithrombin. This is
the un-competitive inhibitors as it can bind the thrombin after it have interacted with the fibrinogen.
Antithrombin can bind the thrombin in 2 specific AAS (arginine and serine) far from the active site; the super
complex thrombin-enzyme-fibrinogen-substrate-antithrombin is useful to stop the function of thrombin.
75
Metabolism Metabolism is the set of life-sustaining chemical transformations within the cells of living organisms; it
includes reactions allow organisms to grow, to maintain their structures and to respond to their
environments. It is referred to all chemical reactions present in living organisms, including digestion and
substances transport into or between different cells. Is due by an huge number of chemical reaction able to
change the initial compound into any others. It could be used to transform compound or produce energy.
Metabolism is divided into two categories:
a. Anabolism is the use of energy to construct components of cells such as proteins and nucleic acids;
the energy is used in order to create new chemical bounds.
b. Catabolism is the degradation of organic compounds to produce energy for cellular life. For example
one catabolic pathway is glycogenolysis.
Anabolism and catabolism form a sort of cycle: compounds and
energy (ATP) produced by catabolism can be used is anabolism in
order to form new biomolecules.
Both anabolic and catabolic reactions are catalysed by enzymes in
order to increase the reaction rate; they often require dietary
minerals, vitamins and other cofactors. Moreover, the regulate
metabolic pathways in response to changes in the cell's environment
or to signals from other cells.
Organism are open system in contact with the external environment. For each biomolecule, there is a specific
metabolic pathway able to digest compound into simpler biomolecules useful for our body.
Meat is a source of protein and therefore AAs; meat protein and endogenous proteins are both destroyed by
the urea cycle. Regarding glucose, the main external source is in carbohydrates and sugar and used in the
glycolysis in order to produce energy or in the glycogen synthesis. The first catabolic pathway involved for
the production of ATP is the Krebs (TCA) cycle, able to produce an huge amount of energy; the starting point
of TCA is the first product of glycolysis. Fats are useful in order to obtain fatty acid, phospholipids
components.
Thermodynamics of metabolism Metabolic pathway responds to specific thermodynamic law, in particular the third. Therefore every
metabolic pathway has a specific Free Gibbs Energy determining the reaction spontaneity: exergonic reaction
have a negative Free Gibbs Energy so the final compound has lower energy than the initial one. It depends
on the concentration of initial substrate and final products and the experimental condition (fixed).
All metabolic reactions are catalysed by specific enzymes and they influence the metabolic pathway. As well
as reversable (near the equilibrium) and irreversible reactions exist, so do the enzymes: reversable enzymes
are able to transform the substrate into the product and viceversa.
76
Such reversable reaction prevent the flux of the pathway due to a sort of equilibrium, while this vicious cycle
is disrupted if there is at least one irreversible reaction (∆𝐺 ≠ 0); actually, the reversible reaction would be
possible, but is isn’t catalyse by the enzyme.
Through alternative cycles, the latter products could be reversed into substrates.
Flux control Metabolic flux is a sort of metabolic rate and is due to the reaction rate of only irreversible reaction as they
are characterized by only one specific reaction rate.
Normally, the flux rate is similar to the slowest reaction present in the metabolic pathway; on the contrary,
there is a sort of independence from the substrate concentration.
In order to control the pathway, it is necessary to control the enzymes; for this reason, metabolism flux can
be controlled by the same mechanism of enzymes.
a. The genetic modulation (long term control) requires long time in order to be express and to be stop
as well and therefore is not commonly used as the cell requires a short and fast modulation.
b. The metabolic pathway is generally controlled by short term controls: allosteric control, covalent
modification and substrate cycles;
Allosteric control: there are 2 different possibilities:
o Product inhibition: the product is able to
inhibit the enzyme involved in the catalysis
(the first product B inhibits E1).
o Feedback inhibition: the final product of the
entire metabolic pathway is able to inhibit
the first enzyme (P inhibits E1).
Covalent modulation: covalent modifications (phosphorylation/dephosphorylation,
glycosylation…) on enzyme can modify enzymatic activity.
Substrate cycle: considering a metabolic pathway that transform B into C (through the enzyme
E1) and then into D, when the cell doesn’t need D anymore, another enzyme E2 is activated and
this transforms C into B.
ADENOSINE TRIPHOSPHATE (ATP)
ATP is the most important energetic molecule. It is composed by 1
nitrogenous base (adenine), 1 sugar (ribose) and 3 phosphate groups,
involved in the release of energy as they are linked each other through
an high energetic chemical bound called phosphoanhydride bond.
ATP is an energetic molecule because the hydrolysis of such phosphate
groups releases an high amount of energy which could be used by other
biomolecules.
77
There are 2 possible hydrolysis of ATP:
a. ADP is formed by the release of 1 P group
and this creates an high amount of energy;
b. AMP is formed by the release of two P
groups produces just a little increment comparing
with ADP; this 2 P groups remains bond together in
a molecule called inorganic pyrophosphate.
The hydrolysis of ATP is an exergonic reaction: this
means the final product has an higher stability than
the substrate: ADP and AMP are more stable than
ATP and this is the reason why hydrolysis is
favoured.
The hydrolysis of the anhydride bonds is favoured due to different reasons:
ADP or AMP have an higher stability of resonance: one
negative charge (on the O of the P group) could be
delocalised into different atoms. In the ATP the
movement of the electrons is prevented because of the
repulsion between negative charges, while after the
hydrolysis this doesn’t occur.
Moreover, in ADP and AMP there is a minor electrostatic
repulsion thanks to a reduction of negative charges.
Lastly, in ADP and AMP the hydratation layer in higher and this increases the stability.
Values of Gibbs free energy for ATP hydrolysis depend on the overall ionic strength and on the presence of
alkaline metal ions (Mg2+ and Ca2+): these favours ATP hydrolysis by reduction of electrostatic repulsion
neutralizing negative charges on products.
Exergonic metabolic pathway ATP is normally produced during catabolic pathway, but it can also be required during the metabolic pathway.
An example is the glycolysis, which is the catabolic pathway able to transform glucose into wastes and ATP.
During the first reaction of glycolysis, there is the request of energy: the ATP is hydrolysed in order to favour
the first endogenous reaction.
The first step of glycolysis includes 1 molecule of glucose and an ATP transformed into glucose phosphatase,
while latter reactions are exergonic and produce ATPs: the final amount of energy is negative and therefore
the reaction is favoured.
From a kinetic point of
view this isn’t favoured
so it wouldn’t be
possible without one
specific enzyme. The
possibility to use 1
specific enzyme and to
couple reactions transform the first reaction into a thermodynamically ad kinetically favoured reaction.
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The release of energy is not a parameter in order to control the hydrolysis between ADP and AMP as the
energy variation is not very large; therefore, what influence this choice is the other product of the pathway:
the inorganic pyrophosphate. For example, in the activation of AAs (that occurs through ATP), the energy
required could be well produced by the hydrolysis of just one P group. Anyway, it occurs the hydrolysis of 2
P groups in order to form the inorganic pyrophosphate which is divided into 2 inorganic P through the
inorganic pyrophosphatases in an irreversible reaction. In this way, all the pathway becomes irreversible.
ATP turnover The majority of ATP molecules are produced by specific metabolic pathways like Krebs cycle, but there are
other specific reaction involved in maintaining constant the availability of ATP.
ATP production is possible due to 2 different reaction:
a. the most common is the substrate phosphorylation: is an exergonic metabolic pathway which include
the transfer of phosphate group from high energy substrate (phosphocreatine) to ADP.
b. Another type is the oxidative phosphorylation: ADP phosphorylation due to high energy of
transmembrane proton gradient. In this case is possible to transfer the P group through a movement
of charges (electrons).
In the substrate phosphorylation, a substrate (creatine) is involved in the control of ATP concentration,
independently from the metabolic pathway. Creatine is a biomolecule that could also exist in its
phosphorylated form; the creatine kinase is involved in such equilibrium: this enzyme Is able to move the P
group from 1 substrate into another by considering the request of cell.
If the cell doesn’t require energy, the creatine can move 1 P group from ATP to itself in order to obtain the
phosphocreatine, which is a storage of P groups. When the cell requires energy, the phosphocreatine is able
to release the P group in order to create ATP.
As this reaction is a sort of equilibrium, (∆𝐺 ≠ 0.
NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD) NAD is another type of biomolecule connected with energy. It is able to produce ATP by changing its oxidation
state, considering a movement of electrons present inside the membrane of mitochondria. This charges could
be bound to NAD and FAD. NAD could be present oxidized (NAD+) or reduced form (NADH).
A lot of met reaction are able to produce proton ions.
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Glycolysis Glycolysis is the metabolic pathway able to convert glucose into pyruvate releasing energy in the form of ATP
and NADH. The word was born by the couple of glycose (the old term for glucose) and lysis (degradation).
The first molecule is glucose, which is a quite complex molecule,
composed by 6 carbon atoms; at the end of glycolysis, the
glucose is reduced into 2 simpler linear molecules: pyruvate,
which is
composed
by 3 carbon
atoms (the
number of C
atom is
constant).
Glycolysis
occurs in
the cytosol and is formed by 10 reactions, all catalysed by
specific enzymes.
Glycolysis could be divided into 2 different phases: preparatory and pay-off phase. In the preparatory phase
there is a consumption of energy: 2 molecules of ATP are hydrolysed into ADP and the released energy is
used in order to carry out reactions. Nevertheless, considering the whole reaction, from 1 glucose molecule
2 molecules of ATP and 2 molecules of NADH are produced.
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Preparatory phase
STEP1: G – HX -> G6P
Glucose is a cycle formed by 5 carbon atoms and 1 oxygen atom. The first step of the glycolysis is a
phosphorylation: a phosphate group is bound to the sixth carbon atom (the one outside of the cycle) and of
glucose 6-phosphate (G6P) is formed.
The enzyme involved is the hexokinase (HK) (hexo is referred to the cycle).
In order to phosphorylated glucose, is necessary to have a source of phosphate group: ATP.
This step is important in order to maintain low glucose concentration inside the cell (modifying it) and
prevents the release of glucose due by glucose transporters which work following the concentration gradient;
therefore, the phosphorylation favours all the metabolic pathway.
Moreover, such reaction is irreversible: there is only one possible direction of it and this make possible the
control of the pathway‘s velocity (inhibiting or activating the enzyme)
One important cofactor are the Mg ions, which
are present in every step implying ATP. They
stabilize the negative charges present in the
phosphate groups and favour the hydrolysis
and the release of the first phosphate group.
The phosphorylation of glucose by HK occurs in the cytosol, which is an aqueous environment: all enzymes
are able to interact with water therefore there is a competition between glucose and water to interact with
active site. Nevertheless, glucose has a major affinity and the bond glucose-enzyme causes conformational
modification able to increase specificity for substrate and to eliminate water.
STEP 2: G6P <- PGI -> F6P
The second step consist in the transformation of glucose 6-phosphate into fructose 6-phosphate (F6P).
Both molecules are sugar molecules, but have a different number of atoms in the cycle: while glucose is an
exon, fructose is a pentose.
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This kind of reaction is called isomerization and consists is a rearrangement of atoms able to change the type
of sugar; it is catalysed by glucose phosphate isomerase (PGI). The isomerization is possible thanks to the
formation of instable linear intermediates, both created by the simple opening of the cycle.
The change consist in the position of the carboxyl group: in glucose it is the first carbon atom, in fructose the
second. Such group in able to interact with the last C atom and close the cycle.
This reaction is reversable.
STEP 3: F6P - PFK-1 -> F1,6BP
The third step consist in the second phosphorylation, which creates the fructose 1,6-bisphosphate (F1,6BP);
in this reaction the phosphate group in bound to the first carbon atom.
This reaction is irreversible and catalysed by the phosphofructokinase 1 (PFK-1); for this reason, this step
could be controlled by specific modulation.
The Mg ions act as cofactor for ATP.
STEP 4: F1,6BP <- FBP -> G3P + DHAP
In this step the type of molecule changes and the sugar molecule is converted into 2 simpler linear chemical
structure formed by 3 carbon atom. This reaction is reversable and catalysed by the aldolase.
In order to break the bond, the aldolase forms an instable intermate: a linear compound formed by 6 carbon
atoms is divided into 2 linear compound (3 carbon atoms each) through the hydrolysis of the most instable
bond (the blue one).
The products are a ketose and an aldose group: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-
phosphate (G3P)
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STEP 5: DHAP <-> G3P
The glycolysis pathway works only on the glyceraldehyde 3-phosphate, so the last step of the investment
phase is the transformation of the ketone into the aldehyde: the pathway doubles.
The reaction is a reversable isomerization and is catalysed by the triose phosphate isomerase (TIM). The
difference between the product and the reagent is the position of the double bond.
Even if the most of the reactions are reversible, this metabolic pathway follows 1 specific direction thanks to
the fact that, once started, the concentration of the product are always maintained low as they act as the
substrate for the following reaction.
All in all, the first phase of glycolysis is characterized by the
consumption of 2 molecule of ATP involved in the
phosphorylation of specific substrates. The investment phase
starts with on molecule of glucose and finish with 2 molecule
of glyceraldehyde 3-phosphate.
Pay-off phase The pay-off phase produce, for each molecule of aldehyde given, 1 molecule of NADH and 2 of ATP; these
have to be multiply twice. Considering the preparatory phase, the net gain of glycolysis is 2 molecule of NADH
and 2 molecule of ATP as 2 of them have been consumed in the investment phase.
STEP 6: G3P <- GAPDH -> 3BPG
The sixth reaction is reversable and catalysed by the glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
The reaction is a redox: the substrate (G3P) is oxidized and releases electrons and protons which are acquired
by 1 molecule of NAD (which become NADH).
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As result, the glyceraldehyde 3-phosphate is transformed into 1,3-biphosphoglycerate and there is the bound
of one Pi with the oxygen left alone by the hydrogen atom; the bound O-P is called anhydrase bond and has
an high amount of energy and will be hydrolysed in the following step in order to produce energy.
The catalytic
mechanism of GAPDH
explains the reaction.
The catalytic site of
the GAPDH is
characterized by the
presence of 1
cysteine, which is
involved in the
bonding of the
substrate (G3P); this
bond is favoured by
the presence of a
basic AA, which is
able to accept the
proton ions released
by the substrate.
Moreover, the active
site is specific for
NAD+ as well and
therefore the G3P and the NAD are very close: this activates the redox.
After the redox, the substrate is transformed into 1 instable intermediate, which is soon phosphorylated into
3BPG. After this, both the substrate and the NADH are released.
STEP 7: 1,3-BPG <- PGK -> 3PG
In the seventh step there is the first production of an ATP molecule due by the phosphorylation of the
previous anhydrase bond. This reaction is reversable and is catalyse by the phosphoglycerate kinase (PGK).
At the end of the reaction, 3-phosphoglycerate (3PG) and 1 molecule of ATP are produced.
Due to the presence of ATP, there is the presence of Mg ions.
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From a thermodynamic point of view, the hydrolysis cause a negative Free Gibbs Energy. This is essentiale
for the spontaneity of the previous reaction, where the formation of a covalent bond have required energy
(endergonic reactions are not spontaneous); nevertheless, the couple of the sixth and the seventh reactions
increase the spontaneity obtaining a final spontaneous reaction.
STEP 8: 3PG <- PGM -> 2PG
After the first release of ATP, there are some reactions aimed to the formation of another anhydrase bond
in order to phosphorylate an ADP into ATP.
The first reaction (eighth) is the move of a phosphate group from the third carbon atom to the second.
This is catalysed by the phosphoglycerate mutase (PGM): the phosphate group bond to the third carbon atom
is transferred to the second one.
This is possible due to a specific active site
of the PGM. PGM could be defined as a
phosphoenzyme as it contains a
phosphorylated AA; this group is able to
bind the substrate forming an anhydrase
bond.
The intermediate formed is instable as it
contains 2 phosphate groups (both
negatively charged). For this reason, it
suddenly release the less stable phosphate
group (the one which requires lower energy
to be disrupted): the one bound to the third
carbon atom. This is bound again to the enzyme.
Eventually, the product contains just the phosphate group linked to the second carbon atom, which has an
higher energy than the previous one. Therefore, even if the chemical structure doesn’t’ change a lot, the
amount of energy largely increase.
STEP 9: 2PG <- ENO -> PEP
The energy of the phosphate group could be increased by performing another reaction catalysed by the
enolase and able to produce a double covalent bond in the carbon atom. This reaction could be defined as a
condensation because one hydrogen atom and one hydroxyl group form 1 molecule of water.
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2 Mg ions are present as cofactors as the enzyme is able to interact with them: the first is bond in the active
site, while the second is involved in the stabilization of the oxygen negative charge.
STEP 10: PEP – PK -> pyruvate In the last step it occurs the hydrolysis of the anhydrase bond; it is catalysed by the pyruvate kinase (PK) and
it’s irreversible.
Anyway, the major amount of energy of the pay-off phase isn’t given by this hydrolysis, but from the second
part of the reaction: the tautomerization. In this reaction, a movement of electrons transfer the double bond
from the C-C to the C-O and this largely increases the stability.
All step of pay-off phase have to be considered double
Energy evaluation of glycolysis
The glycolysis products are:
ATP: initial investment of 2ATP and synthesis of 4ATP; therefore the net gain is of 2ATP to use in
cellular metabolism
NADH: production of 2NADH: transporter of energy, released through oxidation reaction
Pyruvate: 2 pyruvate molecules that could be transformed into:
o Lactate or CO2 and ethanol in anaerobic conditions
o CO2 and water through the citric acid cycle in aerobic conditions.
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Glycolysis regulation Glycolysis is formed by 10 chemical reactions, but only the enzyme able to catalyse irreversible reaction can
be controlled (3: step 1, step 3 and step 10).
Considering the first step, hexokinase is not a point of regulation as it is involved in another metabolic
pathways (glycogen synthesis).
Considering the last step, pyruvate kinase is not the principal point of regulation as it’s the last enzyme: if the
cell doesn’t require energy, it prefers to stop the pathway at its start.
Therefore the most important point of regulation is the possibility to inhibit phosphofructokinase as its in the
first phase. The inhibition is possible through allosteric modulation and substrate cycle.
Allosteric modulation Allosteric modulation is only possible in complex enzymes which can exist both in T and R state. PFK-1 is a
quite large tetrameric allosteric enzyme, formed by 2 subunits. Each subunits consist in 2 domains containing:
one active site, able to bind ATP and one allosteric site, able to bind ATP, fructose-1,6-biphosphate and AMP.
These are allosteric modulators: an high concentration of ATP means that the cell doesn’t require energy
anymore (R→T), while AMP is an allosteric activator (T→R); also fructose1,6-biphosphate is an activator as
it is produced by the enzyme itself: this is called feedforward.
Substrate cycle control Phosphofructokinase is able to transform the F6P into the F1,6BP; at the same time, fructose diphosphatase
is able to catalyse the opposite reaction.
Both enzymes are present in the cytosol, but their activity could be very different considering the energy
requested by the cell. When it
needs ATP, the activity of
fructose diphosphatase in
inhibited and the majority of
fructose 6 phosphate is
converted into fructose 1,6-
biphosphatase and them into
GAP and DHAP; nevertheless, a
few percentage of FBP is
normally reconverted into F6P.
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Glycogen Glycogen is a long term glucose storage,
which is it’s unit.
It is an energetic source important in order
to produce energy for metabolism and for
the production of new biomolecules. It is
present in muscles (in order to quickly have
energy in them) and liver (as it is involved in
the production of a lot of enzyme).
Glycogen is a polimer as it’s formed by an high number of units.
It contains a branched linear structure, which is functional to reduce its
volume; the number of branches depends on the concentration of
glucose.
The linear structure is composed by covalent binding between glucose molecules, which are called glycosidic.
There are 2 different glycosidic binds, depending on
the position of them:
a. a(1→4) glycosidic bonds occurs between the
first and the fourth carbon atom and it’s
useful in order to form the linear strucure.
b. a(1→6) glycosidic bonds occurs between the
first and the sixth (outseide the cycle) carbon
atom and it’s useful in order to form the
branches. Their number is proportionale to
the number of glucose molecules.
Glycogen is involved in 2 different metabolic pathway: one catabolic, glycogenolysis, and one anabolic,
glycogenosynthesis.
Glycogenolysis Glycogenolysis is the breakdown of glycogen (n) into glucose-1-phosphate and glycogen(n-1).
It’s perforemd by one
specific enzyme and –
as the release of energy
must be quickly – has a
quiete simple
mechanism.
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The enzyme is the glycogene phosphorylase, which is able to perform 2 different chemical reactions on the
linear structure: the hydrolysis of alpha 1-4 glycosydic bond and the phosphorylation of glucose. This occurs
in position 1 because this was previosly involved in the hydrolysis. The glycenolysis is specific for this reactions
and therefore it stops before the branches; moreover it only works in the forward direction when the
concentration of inorganic phosphate is higher than that of glucose-1-phosphate.
Catalytic mechanism
The phosphorylation is due to the presence in the catalytic site of the phosphate group (so the enzyme can
be defined as a phosphoenzyme): this interact with the inorgani phosphate bound to the terminal glucose.
The first step is the formation of H bonding between the phosphate group of the enzyme and the Pi bound
to the gluose in the final step; this destabilizes the last glucose present inside the glycogen as it destabilizes
the alpha 1→ 4 bond.
The protonated oxygen of glycogen represent a good leaving point and the glycogen chain is separated from
the terminal molecule, forming one free glucose and a secondary carbocation, called carbocation
intermediate. Therefore the inorganic phosphate binds to the carbocation generating one glucose 1-
phosphate and this strong bond destabilize the H bond between the 2 phosphate group; eventually, there is
the release of a glycogen chain shortened by one glucose molecule.
Debranching enzymes Debrancing enzymes convert the branched glycogen
structure into a linear one, which can be degradated by
glycogen phosphorylase; they are able to hydrolyse four
molecole present in the branche.
The first debranching enzyme is the glucosyltransferase,
which transfer 3 glucose molecule from the branch to the
linear chain, where they can be hydrolysed by the
phosphorylase.
The fourth glucose molecule is connected through the
alpha(1→6) glycosydic bond, which is hydrolysed by a
specific enzym called glucosidase.
Such enzyme are able to produce glucose 1 phosphate.
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Phosphoglucomutase The last inzyme involved in the glycogenolysis is
the phosphoglucomutase, which is able to move
the phospate group from position 1 to position
6 of the released glucose and viceversa.
In the latter case the enzyme is also involved in the glicogenosynthesis.This common enzyme between 2
metabolic pathways permit to reach an equilibrium between the
possiblity to store and produce energy.
Therefore glucose 6-phosphate could be produce both by the direct
phosphorylation in glycolyssi and by the glycgenoysis. G6P
monomers are able:
to continue the glycolysis pathway
to produce NADPH and 5-carbon sugars through pentose
phosphate pathway
to enter in final step of the gluconeogenesis pathway in the
liver and kidney
It represent the bond between glycolysis and glycogenolysis, which
aliment the first. This is the reason why the first step of glycolysis
can’t be controlled for its modulation: it must supply the substrate
for phosphoglucomutase.
Glycogenesis Glycogenesis is the process of glycogen synthesis,
in which glucose molecules are added to chains
of glycogen for storage. It is activated during rest
periods in the liver and by insulin in response to
high glucose levels.
The glycogenesis starts when glucose is
transformed into G6P by hexokinase and then
transformed into G1P by phosphoglucomutase.
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UDP-glucose pyrophosphorylase G1P can be activated in order to bound to glycogen; such activation is
performed by UDP-glucose pyrophosphorylase; UTP is an ATP-like
molecule, indeed it contains 3 phosphate group, ribose and a
(different) nitrogenous base.
In this reaction there is the hydrolysis of the glycoside bond, through
the release of 2 phosphate group from UTP (which becomes UMP):
UDP-glucose is formed. UDPG has some chemical features useful to
bind to glycogen.
This activation is reversable because the Free Gibbs Energy is near 0;
this means that in the presence of 1 specific product, the reaction
could occur backward.
Pyrophosphate is the key-product of the reaction: it is suddenly
irreversibly transformed into 2 molecules of inorganic phosphate by the
inorganic pyrophosphatase.
This is the most important function of ATP-like biomolecules: their hydrolysation can, make reactions
irreversible.
Glycogen synthase Glycogen synthase takes short
polymers of glucose and converts
them into long polymers linking one by
one glucose residue to the polymeric
glycogen chain for storage.
At this point, the unit of glucose is
ready in order to be bound to the
glycogen chain. One specific enzyme
called glycogen synthase is useful in
order to catalyse the formation of
alpha(1→4) glyosidic bond, while
another is involved in the formation of
alpha(1 → 6) bond.
Glycogen can interact with the activated glucose (UDGP) in order to increase its length; this causes the release
of UDP and determines the reaction as exergonic.
Consequently, the UDP is phosphorylate into UTP through 1 ATP.
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The glycogen synthase is the most important enzyme so it’s important to control it. For this reason, the
modulation is quite complex and could occurs both by through allosteric and covalent modulation in order
to increase the possibility to activate or inhibit such enzyme.
The enzyme could be phosphorylated by one specific kinase into the inactive B form or dephosphorylated
into A form. Moreover, both glycogen synthase and the kinase could be modulated by specific modulators.
Possible modulators could be the product of glycogenesis (ADP, Pi, protein kinases) or glycogenolysis (ATP,
G6P).
Glycogenin Glycogen can’t be produced ex-novo, so glycogenin acts as a primer by polymerizing the first few glucose
molecules, after which glycogen synthase takes over. The enzyme involved in the formation of the first
alpha(1→4)glyosidic bond is called
Branching enzyme The last step of glycogenesis is the possibility to
synthetize branches (alpha(1→ 6)glyosidic bond).
In this step there is just one enzyme involved: the
amylo-alpha(1:4)→a(1:6) transglycosylase.
It recognizes a specific chain of glucose molecule
and form a new side chain every 10 to 14 units from
the first branched chain. It has a high specificity,
both for the number of glucose units transferred (7)
and for the position to which they are added (4).
As this enzyme need 7 glucose molecules, the
primer formed by glycogenin and glucose is formed
by 7 glucose. The specific number is due to the
sterical hindrance compatible with debranches
enzyme, which wouldn’t be able to hydrolyse the binds.
Hormonal control of glycogen metabolism The glycogenolysis and the glycogenesis are controlled by specific biomolecules, such as hormones secreted
by specific organs or glands.
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E.g. insulin is synthesized inside pancreas and is secreted when the concentration of sugar in blood increase;
it is bound to specific receptors present in liver and muscles on the cellular membrane. This interaction starts
a cascade which ends with the activation of glycogen synthase.
On the contrary the glycogen degradation is activated by epinephrine, secreted by adrenal glands or by
glucagon, secreted by pancreas.
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Krebs cycle or citric acid cycle (TCA, Tricarboxylic acid cycle)
Glycolysis is not the most effective way to obtain energy as the production is of only 2 ATP molecules;
otherwise, Krebs cycle is specific for energy production because the quite simple degradation of acetyl CoA
forms 10 molecules of ATP.
The name Krebs cycle derives from the scientist who first discovered such metabolic pathway; it can also be
called citric acid cycle or TCA, because of its first products: citrate is a 3carboyl acid, characterized by 3
carboxyl groups.
This metabolic pathway occurs in the mitochondria matrix.
The degradation of the first substrate (acetyl CoA) produces 1
GTP molecule and NADHs and FADHs, involved in the redox
reactions. Another important product is carbon dioxide, as it’s a
regular waste present inside our body and cells are have specific
reactions in order to eliminate this non toxic waste.
Acetyl-CoA synthesis The first substrate of the Krebs cycle is the acetyl CoA, which has
an acetyl group, derived from one specific substrate and is
involved in Krebs degradation; moreover, the CO2 is due to the
degradation of this group.
The acetyl group is bound to the coenzyme A through a thioester
bond, which is highly energetic.
The source of acetyl CoA could be the pyruvate produced during the
glycolysis, indeed it has an acetyl group which could be found in the
acetyl CoA.
The binding between such chemical group and coenzyme A is
catalysed by one specific and complex enzyme called pyruvate
dehydrogenase. This enzyme is composed by a high number of
subunits and so it’s called enzymatic complex.
The reaction consists in the oxidation of the pyruvate and the
reduction of NAD into NADH; this release 1 molecule of CO2.
This kind of reaction occurs 4 time in the Krebs cycle and
all of these are catalysed by pyruvate dehydrogenase.
Pyruvate dehydrogenase is formed by a high number of
different subunits, which can be divided in 3 main parts:
pyruvate dehydrogenase E1, dihydrolipoyl transacetylase
E2 and dihydrolipoyl dehydrogenase E3.
94
the pyruvates bound to the E1 units, which
is characterized by a specific binding site
formed by TTP, able to bind directly to the
carbonyl group and this binding transform it
into 1 hydroxyl group. Moreover, there is the
releasing of the first product: CO2.
After this interaction the E1 is very close to
E2.
E2 has a cyclic group formed by a disulphide
bridges which can bind the acetyl group and
pick it up from E2.
The binding of acetyl group causes the
opening of cycle causing the break of
disulphide group; this favour the interaction
with coenzyme A.
Accordingly, the acetyl group is transferred
from E2 to coenzyme A in order to form the
second product: the acetyl-CoA which is involved in the Krebs cycle.
The following steps are all aimed to the restore of the enzyme in its initial state.
The oxidation of thiol group restores the sulfuric cycle and the FAD is reduced to FADH.
The last step is the transfer of such electrons from FADH to NAD: FADH is oxidized and the NAD is
reduced to NADH.
This catalytic mechanism is cyclic and produces carbon dioxide, acetyl coenzyme A and NADH.
Krebs cycle
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Krebs cycle is composed by 10 reactions and is continuously activated as each substrate is constantly
produced. The acetyl CoA is the first substrate and it represent the link with the catabolism of
carbohydrate, fat and protein.
The energetic molecules are
produced in 3 different forms: 1
GTP, which is similar to ATP and
corresponds to 1 molecule of ATP;
3 NADH which corresponds to 1,5
ATP and 3 NADH which correspond
to 7,5 ATPs.
In conclusion, the Krebs cycle
produces 10 molecules of ATP.
The first reaction of Krebs
cycle involves acetyl CoA
and the final product of
the previous cycle:
oxaloacetate.
Therefore, as the carbon
dioxide is formed by the
carbon atoms present in
the oxaloacetate and not
from those in the acetyl
CoA, the production of
CO2 occurs only from the
second cycle; in the first
one the first acetyl CoA
forms products which
compose oxaloacetate.
STEP 1 The fist reaction is a condensation which produces citric acid (citrate) – a three carboxyl acid – and is
catalysed by citrate synthase. This enzyme is specific for acetyl CoA and oxaloacetate, but only in a specific
order: the bind of oxaloacetate increases the specificity for acetyl CoA.
The low distance between the 2 substrates is useful in order to produce the 1 instable intermediate called
citroyl-CoA intermediate, which contains an instable highly energetic thioester bound due to the high
sterical hindrance of the coenzyme A.
96
The hydrolysis of such bind occurs and there is the release of the Coenzyme A. This hydrolysis stabilize the
formation of a six-carbon citrate.
STEP 2 The second reaction is an isomerization and changes the position of the hydroxyl group from the third to
the fourth carbon atom; this produces the isocitrate, which is more stable because of the increased
distance between the hydroxyl and the carbonyl group.
Step 2 is catalysed by aconitase which works in 2 steps and involves 1 water molecule: in the first step there
is the elimination of 1 water molecule due to the interaction of hydroxyl group and one H. This contributes
to the formation of a double chemical bond. The intermediate (cis-aconitate) is instable but is useful in
order to change the conformation of the molecule.
The possibility to perform such chemical bond causes a flip of all chemical groups from citrate to isocitrate.
Then, aconitase catalyse the addition of water to the double bind, favouring the attachment of carboxyl
group to the fourth atom.
STEP3 The third step is the first in which energy is produced after 3 preparatory steps: an oxidation produces a
molecule of NADH.
This reaction is catalysed by isocitrate dehydrogenase and involves the carboxyl group; there is the release
of the first carbon dioxide molecule and the reduction of NAD into NADH.
The enzyme works in a 2-step process: the first is the redox and produce and instable intermediate:
oxalsuccinate; secondly, there is the decarboxylation of the beta-carboxyl group to a ketone, the alpha-
ketoglutarate.
STEP 4 The fourth step is important in order to forma another energetic chemical bonding: the alpha-ketoglutarate
is bound to molecule of coenzyme A in order to from succinyl CoA. It can be defined as an oxidative
decarboxylation catalysed by the alpha-ketoglutarate dehydrogenase (which act as the pyruvate
dehydrogenase). The subunit of the enzyme are: E1 oxoglutarate dehydrogenase, E2 dihydrolipoyl.
Succinyl CoA has just one carboxyl group while the keto group is involved in the binding with coenzyme A
with an thioester chemical bond.
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STEP5 In the fifth step, the succinyl CcoA is transformed into succinate through the release of coenzyme A; the
hydrolysis of such thioester group phosphorylates one molecule of GDP into GTP.
The reaction is catalysed by the succinyl-CoA synthetase, which has an histidine AA involved in the
phosphorylation.
The succinyl CoA can interact with an inorganic phosphate
thanks to the enzyme, which binds both compound in its
active site.
This interaction causes the displacement of CoA from
succinyl CoA by hydrolysation of the thioester bind and
the formation of the orthophosphoric chemical bond.
This intermediate isn’t stable due to the number of close
negative charges and this causes the release of the
inorganic phosphate, which remains bound to the N of the
histidine cycle. Successively, the Pi interact with the GDP.
STEP 6 The aim of the sixth and seventh reactions is to prepare the last oxidation; this transform succinate into
fumarate through the formation of a double chemical bond. It can be defined as an oxidation because the
new chemical bound is formed after the release of 2 carbon atom, which reduce FAD to FADH.
The elimination of such H atom is performed through the succinate dehydrogenase.
This enzyme is physically bound on the membrane of mitochondria as its also involved in the electron
transport chain.
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STEP 7 step 7 is a sort of hydration which removes the double chemical bond through the interaction with one
water molecule.
This reaction is catalysed by fumarase, which is present in more than one metabolic pathway (catabolism of
protein and AA) and can exist in two different form: mitochondrial or cytosolic.
STEP 8 In the last step there is the oxidation of NAD into NADH, following the release of hydrogen atoms from the
carboxyl group. Oxaloacetate is formed.
The enzyme belongs to the dehydrogenase enzyme: malate dehydrogenase.
Such oxaloacetate starts a new cycle.
Krebs cycle regulation
Being the most important energetic pathway, Krebs cycle must be accurately regulated; the modulation
could occur only in those reaction whose Free Gibbs Energy is lower than 0.
From the table is possible to see that in
physiological condition, just 3 enzymes catalyse
irreversible reactions: Citrate synthase, Isocitrate dehydrogenase
and Alpha-ketoglutarate dehydrogenase.
Overall, these three enzymes could be modulated through:
covalent control (phosphorylation),
product inhibition: the product of 1 reaction can inhibit
the enzyme of the previous reaction,
allosteric modulation: some enzyme can function only if
specific cofactors are present, e.g. Ca.
Enzyme ∆𝐺0′ ∆𝐺
Citrate synthase −31,5 < 0
Aconitase ~5 ~0
Isocitrate dehydrogenase −21 < 0
Alpha-ketoglutarate dehydrogenase −33 < 0
Succinyl-CoA synthetase −2,1 ~0
Succinic dehydrogenase +6 ~0
Fumarase −3,4 ~0
Malate dehydrogenase +29,7 ~0
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Krebs pathway is related to other
metabolic pathways:
glycolysis produces
pyruvate, which can be
transformed into acetyl CoA
lipids metabolism produces
acetyl CoA
proteins metabolism can
produce various intermediates:
o aspartate, tyrosine and
phenylalanine produce fumarate
o isoleucine, methionine and
valine produce succinyl CoA
o glutamate produces alpha-
ketoglutarate
o alanine produces pyruvate
which is transformed into acetyl
CoA.
The fact that proteins mainly enter in the last step of Krebs cycle makes clear that they do not have a key-
role in the production of energy.
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Lipids: triglycerides Lipids are the main producers of acetyl CoA and, consequently, the major source of ATP.
Triglyceride is an ester derived from glycerol (primary
alcohol) and 3 fatty acids, a similar structure of the
phospholipids; they are defined as hydrophobic as they miss
charged groups; for they are instable in an aqueous
environment they are stored in adipose tissue or in the liver.
Triglycerides metabolism One source of triglycerides is lipidic food;
their degradation starts inside the stomach
and not inside the mouth, where
carbohydrates are first deteriorated. Inside
the stomach there is a connection with liver
which can produce enzyme useful for the lipid
degradation; the degradation continues
inside the intestine, where there is the major
achievement of pancreatic lipase.
Lipase are only able to digest them after the
interaction with colipase formed by the
pancreas; together they form a complex
which hydrolyse ester bindings between
glycerol and fatty acids forming monoglycerides and free fatty acids.
These compounds can cross the enterocytes cellular membrane, which only absorb simple lipids and
monoglycerides; after the cellular internalization an opposite reaction is catalysed: triglycerides are formed
again from fatty acids and monoglycerides and packaged into lipoproteins which are released into the
capillaries of the lymph system and then into the blood in order to reach all peripheral tissues. Eventually,
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they bind to the membranes of hepatocytes, adipocytes or muscle fibres, where they are either stored or
oxidized for energy.
There are different types of lipoproteins, but chylomicrons are those which
transport lipids absorbed from the intestine to adipose, cardiac and skeletal
muscle tissues, whose which request the major amount of ATP; they are
produced by enterocytes. Their structure is like the liposomes’ one: one
layer of phospholipids with hydrophilic heads on the surface transport
triglycerides as a cargo in the blood stream or lymphatic system. The
hydrophilic layer allows them to create an interaction with the cellular
membrane, while internal fatty acids bind triglycerides.
Different types of chylomicrons exist, considering their composition and the embedded protein:
a. nascent chylomicrons are formed by a
large portion of triglycerides core; they can
flow in the lymphatic system and blood
flux and reach every tissue to release their
cargo;
b. chylomicron remnants have lost mostly of
triacylglycerol core and are taken up by the
liver, transferring dietary fat also to the
liver.
Hepatocytes have different functions: first of all, they
store triglycerides but also they create triacylglycerols via
de novo synthesis. This latter function is fulfilled
assembling proteins in order to create very low-density
lipoproteins (VLDL) which are released into the
bloodstream by the liver and there they are absorbed by
peripheral tissues and release triglycerides. This modify
them into low-density lipoproteins (LSL), able to continue
the transport of triglycerides and go back to the liver,
where not immediately useful triglycerides are stored.
Eventually, high-density lipoproteins (HDL) collect fats
(phospholipids, molecules cholesterol, triglycerides,
etc.) from the body's cells/tissues and take it back to
the liver, reducing their bloodstream concentration
(periphery → liver).
This function is essential: an high content of fats in
the blood flux they are store inside the blood vessel
and reduce their lumen and damage their membrane
forming a plaque due by the action of platelets during
the coagulation cascade; this phenomenon causes
ischemia due by the reduction of blood in specific
tissues (brain, heart).
While hepatocytes’ function is the same to the chylomicrons’ one, they differ because of their inner volume
(lower), the starting point (VLDL start from the liver) and the composition of the embedded proteins.
The liver can be defined as a secondary source of triglycerides as it produces a high number of enzymes and
therefore here occurs a lot of anabolic pathways which require energy.
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Eventually, in the lipid metabolism the liver is important in order to control the request of energy from
peripherical tissue as it produces specific lipoproteins which transfer triglycerides (VLDL) into different areas.
Fatty acids degradation The component of triglycerides which produces energy are the fatty acid chains and, as they are three, the
production of ATP is maximised. They are broken down into their metabolites with a final production of acetyl
CoA.
Catabolism of fatty acids could be divided into 3 major steps, which occur in different tissues:
1. lipolysis occurs inside the adipose tissue, where triglycerides interact each other in order to increase
their density. After the lipolysis phase, fatty acid chains are released and transported in the cytosol.
2. In the cytosol there is the activation of fatty acids: they are bound to a molecule of coenzyme A and
become fatty acid CoAs.
3. The activated fatty acids are transported by carnitine system into mitochondria where their
degradation occurs.
The degradation consists in an oxidation: the result is a high number of acetyl CoA which can enter
in the Krebs cycle. This is the link between the catabolism of lipid and the production of energy in the
Krebs cycle.
The first reaction is the activation of fatty acid and is catalysed by thiokinase presents in the cytosol: it
performs a new thioester bound between the fatty acyl chain and CoA. The formation of this new chemical
bond requires energy (ATP).
This ATP consumption is relegated to this first activation as for the successive reaction the energetic molecule
derives from the last step.
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The activated fatty acids are transported into the mitochondria in order to perform the real degradation:
from a 20 carbon atoms fatty acid, 10 molecule of
acetyl CoA (2 carbon atoms) are produced;
eventually, 10 ATP molecules can be produced from
1 acetyl CoA.
All in all, a triglyceride can produce 300 ATP
molecules.
Such degradation is defined an oxidation as 2 out 3
reaction are redox.
Even if the main product of the degradation are ATP
molecules, also FAD and NAD are reduced.
The first oxidation creates a covalent bond
between alpha e beta carbon.
Then there is an hydration which eliminates
the double chemical bond and introduces an
hydroxyl group; these are potential oxidable group.
Successively, such hydroxyl group is oxidizes
into carbonyl group coupled with the reduction of
NAD into NADH. This redox destabilize the initial
covalent bound (of the alpha atom) as 2 carbonyl
groups are very close.
The last step is the hydrolysis of this covalent
bond and the attachment of a new coenzyme A to
the fatty acid chain with a lower number of carbon
atom.
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Proteolysis Proteolysis is the mechanism of protein degradation into short peptides till single AAs. It is involved in many
metabolic pathways in order to reach the homeostasis: exogenous (food) proteins can be absorb through
proteases in stomach and intestine in order to provide AAs to the organism, it can activate protein during
polypeptide synthesis, it can regulate some physiological and cellular processes, for instance preventing the
accumulation of unwanted or abnormal proteins.
There are 2 process of intracellular protein degradation:
a. Autophagy-lysosomal pathway is non-specific and is
performed through lysosomes which fuse with autophagic
vacuoles and dispense their enzymes into the vesicles,
digesting their content. These enzymes are called cathepsins
and are enhanced by the acidic pH.
After this fusion the product of hydrolysis is released into the
ECM through exocytosis and through vesicles which protect
such hydrolysing molecules from the cytoplastic
environment.
b. Ubiquitin-mediated process is a selective
process. The cell can recognize proteins to
be digest thanks to ubiquitin which binds
the protein that has to be destroyed. The
the polyubiquinated protein is a target for
ATP-dependent protease complex, called
proteasome, an example of enzymatic
complex formed by a high number of units
characterized by specific function. It
recognises the ubiquitin and therefore the
protein to hydrolyse. such hydrolysis
requires energy (ATP dependent).
Proteolysis’ aim is to obtain AAs; such peptides can be used in order to synthase new functional proteins but
is possible also to obtain their complete degradation.
The complete degradation could be performed by 2 type of reaction: deamination is the elimination of the
amino, which group is transformed into ammonia, toxic compound neutralized by cell and eliminated
through the urea cycle. Otherwise, the
degradation occurs through transamination
and the product are used in other pathways.
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Transamination Transaminase is an enzyme able to catalyse a reaction between an amino acid and an alpha-keto acid: the
NH2 group on the AA is exchanged with the carbonyl group of the acid.
For example, glutamate and
oxaloacetate are
neutralised in the liver
producing alpha-
ketoglutarate and
aspartate.
Deamination The oxidative deamination is the
elimination of the amino group through
an enzyme called dehydrogenase.
This coupled with the transformation
into ammonia contributes to oxidize the
OH of AAs to produce the carbonyl
group.
Urea cycle Urea cycle or ornithine cycle is a
cycle of biochemical reactions
occurring in liver for production of
urea from ammonia; urea in an
organic compound normally
eliminated by urine.
The pathways formed only by only 5
reactions: 2 occur in the
mitochondria and the other 3 in the
cytoplasm.
The cycle converts two amino groups
(one from NH4 carbon atom from
Asp) and a carbon atom from HCO3-
into urea consuming 3 ATPs.
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Amino acid degradation The relationship between Krebs cycle and the proteolysis. …
In physiological condition the proteolysis stops with the formation of AAs which are used to form other
proteins; nevertheless, this catabolism is increased in pathological conditions where the cell use proteins in
order to have additional energy; in physiological condition the energy is mainly produced by lipids.
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Conclusions
The glycolysis is the catabolism of glucose which derives from carbohydrates; it also produces energy, but its
mainly product is pyruvate; this can be transformed into acetyl CoA to start a Krebs cycle. When glucose
concentration is too high, this is stored as glycogen, produced through glycogenesis.
The Krebs cycle is the most important catabolic pathway to produce energy and a lot of compound can enter
in this cycle at different points.
Fats have both a catabolic and anabolic pathway: fats are degraded into fatty acids which can be transformed
into fat again (energy storage) and phospholipids (component of cellular membrane).
The catabolism of proteins is important to produce AAs and, in a few percentage, such AAS can be hydrolysed
in order to produce different intermediated of the Krebs cycle; the complete catabolism produces ammonia
which is neutralized through the urea cycle.
