biomolecules -ii.docx

7/27/2019 biomolecules -II.docx

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BIOMOLECULES-II


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Factors affecting enzyme kinetics:

The contact between the enzyme and substrate is the most essential pre-requisite for enzyme

activity. The important factors that influence the velocity of the enzyme reaction are discussed

hereunder.

1. Concentration of substrate:

Increase in the substrate concentration gradually increase the velocity of enzyme reaction

within the limited range of substate levels. A rectangular hyperbola is obtained when velocity

is ploted against the substrate concentration. Three distinct phases of the reaction are

observed in the braph.

a) At low substrate concentration, the velocity of the reaction is directly proportional to the

substrate level.(part A in graph)

b) In the second phase (part B), the substrate concentration is not directly prorportional to

the enzyme activity.

c) In the third and final phase (Part C), the reaction is independent of the subctrate

concentration.


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2. Enzyme concentration:

As the concentration of enzyme is increased, the velocity of the reaction proportionately

increases. In fact, this property of enzyme is made use in determining the serum enzymes forthe diagnosis of diseases. By using a known volume of serum, and keeping all the other

factors (substrate, pH, temperature etc) at the optimum level, the enzyme could be assayed

in the laboratory. The relative concentration of serum enzyme measured through its activity is

used for the diagnosis of diseases.

3. Temperature

Velocity of an enzyme reaction increases with increase in temperature up to a maximum and

then declines. A bell shaped curve is usually observed.

Temperature coefficient oris defined as increase in enzyme velocity when thetemperature is increased by 10C. for a majority of enzymes is 2 between 0 C and 40 C.

increase in temperature results in higher activation energy of the molecule and more

molecular (enzyme and substrate) collision and interaction for the reaction to proceed faster.

The optimum temperature for most of the enzyme is between 40C- 45C. however, a few

enzymes (e.g. venom phosphokinases, muscle adenylate kinase) are active even at 100C.


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some plant enzymes like urease have optimum activity around 60C. this may be due to very

stable structure and conformation of these enzymes.

In general, when the enzymes are exposed to a temperature above 50C, denaturation

leading to derangement in the native(tertiary) structure of the protein and active site are

seen. Majority of the enzymes become inactive at higher temperature(above 70C)

It is worth noting here that the enzymes have been assigned optimal temperatures based on

the laboratory work. These temperatures, however, may have less relevance and biological

significance in the living system.

4. Effect of pH:

Increase in hydrogen ion concentration (pH) considerably influence the enzyme activity and a

bell-shaped curve is normally obtained. Each enzyme has an optimum pH at which the

velocity is maximum. below and above this pH, the enzyme activity is much lower and at

extreme pH, the enzyme becomes totally inactive.

Most of the enzymes of higher organisms show optimum activity around neutral pH (6-8).

There are, however, many exceptions like pepsin (1-2), acid phosphatase (4-5) and alkaline

phosphatase(10-11). Enzymes from fungi and plants are most active in acidic pH(4-6)

Hydrogen ions influence the enzyme activity by altering the ionic charges on the amino acids

(particularly at the active site), substrate, es complex etc.


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5. Effect of product concentration:

The accumulation of reaction products generally decreases the enzyme velocity. For certain

enzymes, the products combine with the active sites of enzyme and form a loose complex

and, thus, inhibit the enzyme activity. In the living system, this type of inhibition is generally

prevented by a quick removal of products formed. The end product inhibition by feed-back

mechanism is discussed later.

6. Inhibitors:

Enzyme inhibitor is defined as a substance which binds with the enzyme and bring about a

decrease in catalytic activity of that enzyme. The inhibitor may be organic or inorganic innature. There are three broad categories of enzyme inhibition.

1. Reversible inhibition

2. Irreversible inhibition

3. Allosteric inhibition.

1. Reversible inhibition:


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The inhibitor binds non-covalently with enzyme and the enzyme inhibition can be

reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into

i. Competitive inhibition

ii. Non-competitive inhibition

iii. Un-competitive inhibition.

i. Competitive inhibition:

The inhibitor (I) which closely resembles the real substrate (S) is regarded as a

substrate analogue. The inhibitor competes with substrate and binds at the active

site of the enzyme but does not undergo any catalysis. As long as the competitive

inhibitor holds the active site, the enzyme is not available for the substrate to bind.

During the reaction. ES and EI complexes are formed as shown below.

The relative concentration of the substrate and inhibitor and their respective affinity

with the enzyme determines the degree of competitive inhibition. The inhibition

could be overcome bye a high substrate concentration. In competitive inhibition,the value increases whereas remains unchanged.

The enzyme succinate dehydrogenase (SDH) is a classical example of competitive

inhibition with succinic acid as its substrate. The compounds, namely, malonic

acid, glutaric acid and oxalic acid have structural similarity with succinic acid and

compete with the substrate for binding at the active site of SDH.

Among the above compounds, malonic acid is the most potent competitive inhibitor

of SDH.

ii. Non-competitive inhibition:

The inhibitor binds at a site other than the active site on the enzyme surface.

This binding impairs the enzyme function. The inhibitor has no structural

resemblance with the substrate. However, there usually exists a strong

affinity for the inhibitor to bind at the second site. Infact he inhibitor goes not

interfere with the enzyme-substrate binding. But the catalysis is prevented,

possibly due to a distortion in the enzyme conformation.

The inhibitor generally binds with enzyme as well as the ES complex. The

overall relation in non-competitive inhibition is represented below.

For non-competitive inhibition, the value is unchanged while is lowered.

Heavy metal ions (Ag, Pb, Hg. Etc.) can non-competitively inhibit the enzymes by

binding with cysteinyl sulfhydril groups. The general reaction for Hg is

Heavy metals can lead to the formation of covalent bonds with carboxyl groups

and histidine, often resulting in irreversible inhibition.


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iii. Un-competitive inhibition:

This is the third class of reversible inhibition which, however is not very common.

In this case, the inhibitor does not bind with enzyme but only binds with enzyme

substrate complex.

Un-competitive inhibitor decreases both and values of the enzyme.

2. irreversible inhibition:

The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible.

These inhibitors are usually toxic substance which may be present naturally or man-

made.

Iodoacetate is an irreversible inhibitor of the enzyme like papain and glyceraldehydes-3-

phosphate dehydrogenase. Iodoacetate combines with sulfhydril(-SH) groups at the

active site of these enzymes and makes the ancativeDiisopropyl fluorophosphates (DFP) is a nerve gas developed by the germans during

Second World War. DFP irreversibly binds with enzymes containing serin at the active

site e.g. serin protease, acetylcholine esterase.

Many organophosphorus insecticides like melathion are toxic to animal(including man) as

they block the activity of acetylcholine esterase(essentially for nerve conduction),

resulting inparalysis of vital body functions.

Penecilline and antibiotics also act as irreversible inhibitors of serins containing enzymes

in bacterial cell wall synthesis.

3. allosteric inhibition

the details of this type of inhiubition are given under allosteric regulation as a part of a

regulation of enzyme activity in the living system.

Structural Biochemistry/Enzyme/Transition state

.

Definition

By definition, the transition state is the transitory of molecular structure in which the molecule is

no longer a substrate but not yet a product. All chemical reactions must go through the

transition state to form a product from a substrate molecule. The transition state is the state

corresponding to the highest energy along the reaction coordinate. It has more free energy in

comparison to the substrate or product; thus, it is the least stable state. The specific form of

the transition state depends on the mechanisms of the particular reaction.


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In the equation S X P, X is the transition state, which is located at the peak of the curve

on the Gibbs free energy graph.

Application to Enzymes
http://en.wikibooks.org/wiki/File:Transitionstatechem114A.jpg


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The energy required in Transition state is lowered by enzyme. However, the energy levels of

initial and final states remain unchanged.

Enzymes are usually proteins that act like catalysts. The enzyme's ability to make the reaction

faster depends on the fact that it stabilizes the transition state. The transition state's energy or,in terms of a reaction, theactivation energyis the minimum energy that is needed to break

certain bonds of the reactants so as to turn them into products. Enzymes decreases activation

energy by shaping itsactive sitesuch that it fits the transition state even better than the

substrate. When the substrate binds, the enzyme may stretch or distort a key bond and

weaken it so that less activation energy is needed to break the bond at the start of the reaction.

In many cases, the transition state of a reaction has a different geometry at the key atom (for

instance, tetrahedral instead of trigonal planar). By optimizing binding of a tetrahedral atom,

the substrate is helped on its way to the transition state and therefore lowers the activation

energy, allowing more molecules to be able to turn into products in a given period of time. The

enzyme stabilizes the transition state through various ways. Some ways an enzyme stabilizes

is to have an environment that is the opposite charge of the transition state, providing a

different pathway,and making it easier for the reactants to be in the right orientation for

reaction.
http://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Activation_energyhttp://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Activation_energyhttp://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Activation_energyhttp://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Active_Sitehttp://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Active_Sitehttp://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Active_Sitehttp://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Active_Sitehttp://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Activation_energy


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Consider the peptide hydrolysis by chrymotypsin as an example.

In a normal peptide hydrolysis reaction without the help of a catalyst, water acts as a

nucleophile to attack the electrophilic carbonyl carbon. The carbon atom being attacked goes

from its initial sp2 state(trigonal planar) to a new sp3 state(tetrahedral) in its transition state.

In the presence of chymotypsin, however, a better nucleophile is used in the form of the

catalytic triad - Asp 102, His 57, Ser 195 side chains. Moreover, the oxyanion hole, which

consists of the backbone -NH- groups of Gly 193 and Ser 195 of the enzyme, have the N-H

groups positioned in such a way that they will donate strong hydrogen bonds to the substrate's

C=O oxygen, given that the carbon atom is tetrahedral as found in the transition state. This

strains the bonds of the trigonal planar C=O of the original substrate, helping the reaction to

proceed to the transition state. The hydrogen bonds also stabilize the formal negative charge

on the oxygen atoms. In this way, the activation energy of the reaction is lowered and the rate

of reaction thus increases.

Enzyme Inhibition

In 1948, Linus Pauling proposed that transition state analogs should be effective inhibitors of

enzymes. These molecules are mimics of transition states of the substrate of a particular

enzyme reaction. Because they are so similar to the transition states of the substrate, they can

bind to the enzyme, oftentimes much more tightly than the substrate can. The fact that these

transition state analogs bind so tightly to enzymes makes it an effective enzyme inhibitor. The

transition state theory says that the occurrence of enzymatic catalysis is equivalent to an

enzyme binding to the transition state more strongly than it binds to the ground-state reactants.

This theory is based on the two fundamental principles of physical chemistry: Absolute

reaction-rate theory and the thermodynamic cycle. Also, the thermodynamic cycle relating

substrate binding and transition state binding apply elementary transition-state theory to

enzymatic catalysis, which is a restatement of Pauling's description of transition-state binding
http://en.wikibooks.org/wiki/File:Transition.jpg


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in quantitative symbols. He has stated that the catalytic powers of enzymes result from their

highly specific binding of the transition state.

Mechanism of enzyme catalysis:

The information of an enzyme substrate complex (ES) is very crucial for the catalysis to occur. It

is estimated that an enzyme catalyzed reaction proceeds to times faster than a non

catalyzed reaction. It is worthwhile to briefly understand the ways and means through which the

catalytic process take place leading to the product formation. The enhancement in the rate of the

reaction is mainly due to four processes.

1. Acid-base catalysis

2. Substrate strain

3. Covalent catalysis

4. Entropy effect

1. Acid-base catalysis:

Role of acids and bases is quite important in enzymology. At the physiological pH.

Histidine is the most important amino acid, the protonated form of which functions as an

acid and its corresponding conjugate as a base. The other acids are -OH group of tyrosin

-SH group of cystein, and -amino group of lysine. The conjugates of these acids an

carboxyl ions (COO-) function as bases.

Ribonuclease which cleaves phosphodiseter bond in a pyrimidine loci in RNA is a

classical example of the role of acid and base in the catalysis.

2. Substrate strain:

In this, the substrate is strained due to the induced conformation change in the enzyme. It

is also possible that when a substrate binds to the preformed active site, the enzyme

induces a strain to the substrate leads to the formation of product. The concept of

substrate strain explains the role of enzyme in increasing the rate of reaction.

In fact, a combination of the induced fit model with the substrate strain is considered to be

operative in the enzymatic action

During the course of strain induction, the energy level of the substrate is raised, leading to

a transition state.

The mechanism of lysosome (an enzyme of tears, that cleaves 1, 4 glycosidic bond)

action is believed to be due to a combination of substrate strain and acid-base catalysis.

3. Covalent catalysis:

In the covalent catalysis, the negatively charged (nucleophillic) or positively charged

(electrophilic) group is present at the active site of the enzyme. This group attacks the


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substrate that results in the civalent binding of the substrate to the enzyme. In the serine

proteases( so named due to the presence of serin at active site), co valent catalysis along

with acid-basecatalysis occur, e.g. chymotrypsin, trypsin, thrombin etc.

Very often the enzyme bound coenzyme from covalent bonds with the substrate. For

example, in the transaminases, the substrate amino acid forms a Schiffs base with the

pyridoxxal phosphate (coenzyme) bound to enzyme.

Mechanism of chymotrypsin catalyzed hydrolysis of a peptide bond.

Chymotrypsin is a proteolytic enzyme belonging to a broad group of enzymes called serine

proteases that use serine side chain as a reactive nucleophile in the catalyzed reaction.

Chymotrypsin specifically cleaves the peptide bond next to an aromatic side chain and to a

lesser extent the peptide bond next to a hydrophobic side chain like, methionine or

leucine. Since proteases are destructive in nature these are normally synthesized in their

inactive form called zymogen these are suitably activated as per the requirements.

Chymotrypsin is synthesized in the mammalian pancreas as an inactive precursor calledchymotrypsinogen. This precursor is secreted in the intestine where it is activated by

proteolytic cleavage by proteases. The structure and mechanism of action of chymotrypsin

is probably the most extensively studied and understood system. It catalyses the

hydrolysis of the amide (or peptide) bond with the help of a strong nucleophile in the form

of -CH2 OH group of a specific serine residue. The overall hydrolysis is split into two

steps. In the first step an acyl-enzyme ester is formed as an intermediate, which is then

hydrolysed by water in the second step to yield free carboxylic acid and the enzyme is

regenerated.

R NHR'

O

+ H2COH Enzpeptide

CH3 OCH 2

Enz

O

+ R'NH 2

step 2 hydrolysis

R OH

O

H2COH Enz +

step 1

Structure of chymotrypsin:

The precursor of chemotrypsin (i.e. chymotrypsinogen) consists of a 245-residue long

single chain polypeptide, which is held in its natural conformation by five intramolecular


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disulphide linkages. The linkages arebetween cystein residues 1 and 122; 42 and 58; 136

and 201; 168 and 182 and 191 and 220. A schematic structure of chymotrypsinogen

showing the disulphide linkages and cleavages is given in fig.

Chymotrypsin is converted into the active enzyme in a sequence of interesting proteolytic

cleavage. This process is initiated by an enzyme called enterikinase that specifically activates

trypsinogen which in turn produces trypsin. Trypsin then executes the first cleavage ( between

residues Arg-15 and Ile-16) of chymotrypsin. This yields a two chain active enzyme called -

chymotrypsin. It digests itself in terms of a secondary cut between Leu-13 and Ser-14 residues to

generate what is called -chymotrypsin. It is followed by two more cleavages ( between Tyr-146

and between Asp-148 and Ala-149) to give the active -chymotrypsin. -chymotrypsin consists of

three polypeptide chains held in place by three intramolecular and two intermolecular disulphide


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linkages as shown in fig.

Though a number of cleavages take place in the activation process, it is the cleavage after

residue 15 that is most crucial. The amino group of the Ile-16 residue is very important as

it gets into the formation of a salt link with Asp-194 side chain. Such a possibility does not

exist in the zymogen. The salt link in turn introduces conformational changes in thefragment 189-194 whereby the back bone amide bond of Gly-193 residue acquires an

important position from the mechanism p


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proteolytic enzyme, also called Proteinase, any of a group of enzymes that break the long chainlike

molecules of proteins into shorter fragments (peptides) and eventually into their components, aminoacids. Proteolytic enzymes are present in bacteria and plants but are most abundant in animals. In the

stomach,proteinmaterials are attacked initially by the gastric enzymepepsin. When the protein

material is passed to the small intestine, proteins, which are only partially digested in the stomach, are

further attacked by proteolytic enzymes secreted by thepancreas.oint of view.
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