Enzyme

Catalysis and activation energy

- All biochemical reactions that occur in living cells known as metabolism and are catalyzed by enzymes.

- These biochemical reactions can be divided into two types: anabolic and catabolic reactions.

- In anabolic reactions, complex molecules are synthesized from simpler molecules usually with the absorption og energy. They often involve in condensation.

- In catabolic reactions, complex molecules are broken down into simpler molecules. Usually, energy is released during the reactions. They often involve oxidation and hydrolysis.

- All reaction, whether exergonic or endergonic, have an energy barrier known as the energy if activation, or activation energy, which is the energy required to break the existing bonds and begin the reaction.

- In an exergonic reaction, more energy is released compared to what is absorbed.- In an endergonic reaction, more energy is taken in compared to the amount

liberated.

Enzyme as a catalyst

- Enzymes are globular protein with tertiary structure.- Enzymes are organic catalysts. Catalysts make reactions occur at a faster rate

compared to the normal one.- It affects the rate of reaction by lowering the activation energy necessary to

initiate a chemical reaction.- If molecules need less energy to react because the activation barrier is lowered, a

larger fraction of the reactant molecules reacts at any one time.- As a result, the reaction proceeds more quickly.

Properties of enzyme

1. Enzymes are biological catalysts.2. Enzymes are specific for only one particular type of reaction.3. Enzymes are destroyed by high temperature and extreme pH.

4. The activity of enzymes is regulated.

5. Enzymes are coded by DNA in cells.6. Enzymes activity can be affected by temperature, pH, inhibitors, substrate

concentration and enzyme concentration.

7. Enzymes are required in small amounts because they can be used over and over again.

8. Enzymes lower the activation energy of the reactions that are catalysed.9. Their presence does not alter the nature of properties of the end products of the

reaction.10. The catalysed reaction is reversible.

11. Enzyme are not changed or consumed by the reaction.

Mechanism of enzyme actions

- An uncatalysed reaction depends on random collision among reactants.- Because of its ordered structure, an enzyme reduces the reliance on random

events and thereby controls the reaction.- The enzyme accomplishes this by forming an unstable intermediate complex with

the substrate, the substance on which it acts.- When the enzyme-substrate complex or ES complex breaks up, the product is

released; the original enzyme molecule is regenerated and is free to form new ES complex.

- Every enzyme contains one or more active sites, regions to which the substrate binds, to form ES complex.

- The active sites of some enzymes are grooves or cavities in the enzyme molecule.- It has a specific shape into which only one type of substrate will fit. In the same

way, substrates have a surface region that is complementary in term of size, shape, solubility and charge to the active of its enzyme molecules.

- Therefore, enzyme is highly specific.- The mechanism of enzyme can be explained by two hypotheses: lock and key

hypothesis and induced fit hypothesis.

Lock and key hypothesis

- In the lock and key hypothesis, the configuration of the active site is exactly complementary to that of the substrate.

- The substrate binds to the active site to form an ES complex. The enzyme does not form any chemical bond with the substrate.

- The ES complex holds the substrate in a suitable position and lowers the activation energy.

- Products are formed. The product shape is no more complementary to the active site.

- As such, the products leave the enzyme and the enzyme can reused.

Induced fit hypothesis

- In the induced fit hypothesis, the enzyme changes shape upon binding with the substrate. The active site has a shape complementary to that of the substrate only after substrate is bound.

- Both the shape and the charges of the active site force a substrate to enter the enzyme in a specific orientation.

- On binding, both the substrate and active site undergo some changes in shape.

- Chemical reactions take place in the substrate and products are formed. Small rearrangement of chemical groups occurs in both the enzyme and the substrate molecules when the ES complex is formed.

- The product formed can no longer fit properly in the active site and is expelled.- The enzyme reverts to the original configuration and is ready to receive another

substrate.

Factors affecting enzyme activity

Temperature

- At very low temperature, the kinetic energy of the substrates and enzyme molecules are low. It takes a very long time for the substrate and enzyme to bind. Hence, the rate of reaction is very low.

- As the temperature rises, the kinetic energy of the substrate and the enzyme also increases. The number of collisions between the substrate and enzyme increases and the number of ES complex formed increases. For every 10oC rise in temperature, the rate of reaction increases twice.

- At the optimum temperature, the rate of the reaction is maximum.- Above the optimum temperature, the kinetic energy in the substrate and enzyme

increases. The number of collisions between substrate and enzyme also increases but the formation of ES complex decreases. This is because the increased kinetic energy causes the amino acid molecules in the enzyme to vibrate violently.

- At 60oC, all the enzymes are denatured.

pH

- Enzymes function efficiently over a particular pH ranger at a constant temperature. Usually this ranger is narrow.

- The optimum is the pH at which the maximum rate of reaction occurs. This is the most favorable pH value where the enzyme is most active.

- As the pH is lowered, acidity increases and the H+ concentration increases. The increase in H+ disrupts the ionic bonds which help to maintain the shape of the enzyme. Hence, there is an alteration to its active site. If the pH change is extreme, the enzyme is denatured. Restoring the pH to its to optimum level usually restores the rate of reactions/

- Each enzyme has its own optimal pH.

Substrate concentration

- At various substrate concentrations, it is the initial rate of reaction that is taken into account because as soon as the reaction starts, the substrate concentration falls and the product concentration increases, causing the rate of reaction to fall.

- At lower substrate concentration, the rate of reaction is directly proportional to the substrate concentration.

- At higher substrate concentrations, the rate of reaction becomes constant.- It is the limiting factor of enzyme concentration.- Increasing the enzyme concentration will increase the rate of reaction further.

Enzyme concentration

- At lower enzyme concentration, the rate of reaction is directly proportional to the enzyme concentration.

- At higher enzyme concentration the rate of reaction becomes constant. There is no increase as the enzyme concentration increases.

- It is because the substrate concentration is the limiting factor.- To increase the rate, the substrate concentration has to be increased.

Enzyme kinetics

- Enzyme kinetics is the investigation of the rate at which an enzyme works.- It investigates how enzyme binds to substrates and turns them into products.- Michaelis and Menten separated enzyme reations into two stages.

1. In the first step, the substrate binds reversibly to the enzyme, forming the ES complex.

2. In the second stage, the enzyme catalyses the chemical step and releases the products.

- To derive the Michaelis-Menten constant, the following assumptions are made.1. At the maximum velocity of the enzyme [Vmax], all the enzyme active sites are

saturated with substrate and the number of ES complex is the same as the original total amount of enzyme molecules.

2. The total concentration of S is high enough that [Sfree] = [Stotal].3. The reverse reaction of E+P is negligible, so it is ignored.4. The rate of formation of ES is assumed to be the same as the rate of

breakdown to E+S.5. The substrate concentration [S] is many times higher than the total enzyme

concentration [E].6. The total number of active sites also remain constant, that is [total active sites]

= [empty sites] + [occupied sites].

The rate of product formation equals the rate at which ES turns into E+P, which equals k2

times [ES]. This equation isn't helpful, because we don't know ES. We need to solve for ES in terms of the other quantities. This calculation can be greatly simplified by making two reasonable assumptions. First, we assume that the concentration of ES is steady during the time intervals used for enzyme kinetic work. That means that the rate of ES formation equals the rate of ES dissociation (either back to E+S or forward to E+P). Second, we assume that the reverse reaction (formation of ES from E+P) is negligible, because we are working at early time points where the concentration of product is very low.

We also know that the total concentration of enzyme, Etotal, equals ES plus E. So the equation can be rewritten.

Solving for ES:

The velocity of the enzyme reaction therefore is:

Finally, define Vmax (the velocity at maximal concentrations of substrate) as k2 times Etotal, and KM, the Michaelis-Menten constant, as (k2+k-1)/k1. Substituting:

- To determine Km and Vmax, the Lineweaver-Burk plot is useful. This plot is obtained by plotting the reciprocal of V versus the reciprocal of the substrate concentration.

The significance of Km and Vmax

- Different enzymes have different Km values.- Most enzymes have Km values between 10-1 and 10-7 M.- The Km value of an enzyme depends on the substrate and the environmental

condition such as pH and temperature.- A high Km indicates a weak ES binding whereas low Km indicates strong ES

binding.- Vmax shows the turnover number of an enzyme. The turnover number is the

number of substrate molecules an enzyme converts into product molecules per catalytic site in a unit time when the enzyme is fully saturated.

Enzyme cofactor- A cofactor is a non-protein compound that is bound loosely to the enzyme and is

required for biological activity.- Cofactors can be considered "helper molecules" that assist in biochemical

transformations.- Cofactors can also be classified depending on how tightly they bind to an enzyme,

with loosely-bound cofactors termed coenzymes and tightly-bound cofactors termed prosthetic groups.

- An inactive enzyme, without the cofactor is called an apoenzyme, while the complete enzyme with cofactor is the holoenzyme.

1. Coenzyme- Coenzyme is a organic compound that is bound loosely to enzyme and directly

participates in the reaction. It can be detached readily from the enzyme it is bound to.

- Many vitamins are precursor of coenzyme and some vitamins such as vitamin C is a coenzyme itself.

- Coenzymes work by binding to the active side of the enzymes, the side that works in the reaction.

- Since enzymes and coenzymes are nonmetal organic molecules, they bind together by forming covalent bonds. The coenzymes share electrons with the enzymes, rather than lose or gain electrons. When they form this bond, they only help the reaction to occur by carrying and transferring electrons through the reaction.

- Coenzymes do not become integral parts of the enzymatic reaction. Instead, the covalent bonds are broken at the end of the reaction, and the coenzyme returns back to free circulation within the cell until it is used again.

- The NAD CycleNAD---nicotinamide adenine dinucleotide---is a coenzyme that is formed from vitamin B3. It works in several metabolic processes that goes through oxidation---the removal of a hydrogen ion---and reduction, or the gaining of a hydrogen ion. It works as a carrier of hydrogen atoms and transfers them to the end molecules in the enzyme reaction. The NAD coenzyme can be reused by the cell, over and over again.

Prosthetic group- Prosthetic group is organic compound that is bound tightly to enzyme. Unlike

coenzyme, prosthetic group cannot be detached easily from the enzyme unless the enzyme undergoes denaturation.

- Heam is the prosthetic group of the enzyme catalase that catalyses hydrogen peroxide into oxygen and water.

- The heam iron serves as a source or sink of electrons during electron transfer or redox chemistry. In peroxidase reactions, the porphyrin molecule also serves as an electron source.

Metal ions- Metal ions are inorganic cofactor.- Some enzymes are bound tightly to the metal ions such as Zn2+ in

carboxypeptidase and Ca2+ in thrombokinase.- Metal ions help draw electrons away from the substrate molecule making bonds

less stable and easier to break. - Some attach onto the active site of the enzyme to make its shape more efficient

for binding.

Enzyme inhibition- Inhibitor slows down or stops enzyme reaction. There are two types of inhibitor:

competitive and non-competitive.- Non-competitive inhibition can be reversible or irreversible.

Competitive inhibition

- Competitive inhibitor mimics the shape of substrate. It fits temporarily into the active site of an enzyme and prevents the binding of the enzyme with its substrate.

- The competitive inhibitor competes with the substrate for the same active site.- Competitive inhibitors reduce the reaction velocity by lowering the proportion of

enzyme molecules bound to the substrate.- In competitive inhibition, the Km increases and the Vmax remains the same.

Non-competitive inhibition

- In non-competitive inhibition, the non-competitive inhibitor binds to an allosteric site of the enzyme and alters the active sites, resulting in a distorted active site that does not allow substrate to bind to the enzyme.

- The inhibitors reduce the turnover number of the enzyme.- Non-competitive inhibition cannot be overcome by increasing the substrate

concentration.- The Vmax decreases and the Km remains the same, even with non-competitive

inhibitors.

Classification of enzyme

- There are six basic reactions catalysed by enzymes.- Each enzyme is described by a sequence of 4 numbers preceded by the

abbreviation ‘EC’1. EC1-oxidoredutase2. EC2-transferase3. EC3-hydrolase4. EC4-lyase5. EC5-isomerase6. EC6-ligase

- For example, the tripeptide aminopeptidases have the code "EC 3.4.11.4", whose components indicate the following groups of enzymes:

EC 3 enzymes are hydrolases (enzymes that use water to break up some other molecule)

EC 3.4 are hydrolases that act on peptide bonds EC 3.4.11 are those hydrolases that cleave off the amino-terminal amino acid

from a polypeptide EC 3.4.11.4 are those that cleave off the amino-terminal end from a tripeptide

Group Reaction catalyzed Typical reaction Enzyme example(s) with trivial name

EC 1Oxidoreductases

To catalyze oxidation/reduction reactions; transfer of H and O atoms or electrons from one substance to another

AH + B → A + BH (reduced)A + O → AO (oxidized)

Dehydrogenase, oxidase

EC 2Transferases

Transfer of a functional group from one substance to another. The group may be methyl-, acyl-, amino- or phosphate group

AB + C → A + BC Transaminase, kinase

EC 3Hydrolases

Formation of two products from a substrate by hydrolysis

AB + H2O → AOH + BH Lipase, amylase, peptidase

EC 4Lyases

Non-hydrolytic addition or removal of groups from substrates. C-C, C-N, C-O or C-S bonds may be cleaved

RCOCOOH → RCOH + CO2 or [x-A-B-Y] → [A=B + X-Y]

Decarboxylase

EC 5Isomerases

Intramolecule rearrangement, i.e. isomerization changes within a single molecule

AB → BA Isomerase, mutase

EC 6Ligases

Join together two molecules by synthesis of new C-O, C-S, C-N or C-C bonds with simultaneous breakdown of ATP

X + Y+ ATP → XY + ADP + Pi

Synthetase

Technology: enzyme immobilization and biosensing

- Enzymes can be immobilized by binding them to or trapping them in a fixed structure or matrix.

- Advantages1. The stability of the enzyme increases.2. The enzyme can be recovered easily and reused.3. The reaction products are not contaminated by the enzyme.4. The enzyme can be manipulated easily.5. It allows the continuous production of substances.6. No purification of product is needed.7. The cost of operation is lower.8. The operation can be carried out at high temperature or wider pH rangers

without denaturing the enzyme.- Disadvantages

1. The inert matrix may cover some of the active sites.2. The chances of the substrate entering active sites are lower.

Methods of immobilization of enzyme

Entrapment

- The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane.

- It is done in such a way as to retain protein while allowing penetration of substrate. It can be classified into lattice and micro capsule types.

Cross-linking

- Immobilization of enzymes has been achieved by intermolecular cross-linking of the protein, either to other protein molecules or to functional groups on an insoluble support matrix.

- Cross-linking an enzyme to itself is both expensive and insufficient, as some of the protein material will inevitably be acting mainly as a support. This will result in relatively low enzymatic activity.

- Generally, cross-linking is best used in conjunction with one of the other methods. It is used mostly as a means of stabilizing adsorbed enzymes and also for preventing leakage from polyacrylamide gels.

Adsoption

- The carrier-binding method is the oldest immobilization technique for enzymes. - In this method, the amount of enzyme bound to the carrier and the activity after

immobilization depend on the nature of the carrier. The following picture shows how the enzyme is bound to the carrier:

- The selection of the carrier depends on the nature of the enzyme itself, as well as the:

Particle size Surface area Molar ratio of hydrophilic to hydrophobic groups Chemical composition

- In general, an increase in the ratio of hydrophilic groups and in the concentration of bound enzymes, results in a higher activity of the immobilized enzymes.

Production of high fructose syrup (using immobilized enzymes)

- Immobilized enzymes are widely used in the food and drinks industry. One of the uses is to produce high fructose syrup.

- High fructose syrup contains 90% fructose and 10% glucose.- The raw material for the production of high fructose syrup is starch (from corn).- Three main enzymes are used to convert starch to high fructose syrups.

1. Alpha-amylase2. Glucose isomerase3. Glucoamylase

Biosensors

- A biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component.

- It consists of 3 parts:

1. Receptor (uses a biochemical reaction to detect a specific substance)2. Transducer (converts a biochemical signal into an electrical signal)3. Amplifier4. Data processor5. A system to convert the electrical system into a reading or measurement

- One common biosensor is used by diabetics to monitor their blood glucose levels.- The biosensor has a plastic strip with a piece of special filter paper on one end.

- The filter paper contains two immobilized enzymes and a colourless hydrogen donor.

- The enzymes are: glucose oxidase and peroxidase.

- When the paper is dipped in blood containing glucose, glucose is converted to gluconic acid and hydrogen peroxidase by glucose oxidase.

- The peroxidase then catalyses the reaction between the hydrogen peroxide and the colourless hydrogen donor to form a coloured compound.

- The type and intensity of colour indicates the concentration of glucose in the blood.

DNA and protein synthesis

The Griffith experiment

- Griffith used a bacterium called Streptococcus pneumoniae that causes pneumonia in mammals.

- There are two strains of Streptococcus pneumoniae1. The virulent2. The avirulent

- The pathogenic form is also known as the smooth of S form as it has a polysaccharide coat. The S form has the ability to produce toxins.

- The mutant form is called the rough or R form and does not have a polysaccharide coat. The R form does not have the ability to produce toxins.

Avery’ experiment

- Avery isolated and purified different substances from the dead S strain.- Each substance was injected with live R stain cells to determine the transforming

principle.

Treatment OutcomeDNA from S strain + live R strain Mice diesProtein from S strain + live R strain Mice livesRNA from S strain + live R strain Mice livesLipids from S strain + live R strain Mice livesPolysaccharide from S strain + live R strain Mice livesDNA from S strain + DNAse + live R strain Mice lives

- Purified DNA was capable of transforming live R strain to S strain.- When an enzyme that hydrolysed DNA was added to the purified DNA, the R

strain was not transformed. - The conclusion derived was that the hereditary substance was DNA and not

protein.

Gene concept: one gene-one polypeptide hypothesis

- George Beadle and Edward Tatum carried out a series of experiment using the bread mold, Neurospora crassa which reproduces by spores and frows in the bread as a mycelium.

- First, the spores were irradiated with X-rays or UV rays. This procedure increases the frequency of mutations.

- They were then grown in a complete medium that contained all the nutrients necessary for growth.

- One there were enough spores, the spores were then placed in minimal that lacked various substances that the fungus would normally synthesise.

- If the spore did not grow in the minimal medium, then it contained one or more mutations in the gene needed to produce the missing nutrients.

- By adding different nutrients to the minimal medium, the gene that had mutated could be determined.

- For example, an arginine mutant did not grow on a minimal medium, but grew when arginine was added to the minimal medium.

- Beadle and Tatum suggested that irradiating damaged the genes which were involved in the ability to make the compounds needed for the growth of the bread mould.

- Beadle and Tatum were able to isolate three mutant strains that cannot synthesise arginine, they discovered that the mutations wre always located at one of a few chromosomal positions.

- When the chromosomal positions were determined, arg mutations were seen to be clustered in three positions. These clusters correspond to the location of the gene encoding the enzymes that carry out arginie biosynthesis in a multi-step pathway.

- Each of the mutants had a defect in a single enzyme because of a mutation in a single site.

- For example, if mutation occurred at arg-E, then enzyme E would be defective and glumate would not be converted to ornithine.

- Hence, this mutant will not grow in a minimal medium or in a minimal medium with added glutame, but will grow when the minimal medium is added with ornithinem citruline, arginosuccinate or arginine.

- This is because the other enzyme F, G and H are normal and will allow the synthesis of arginine to carry on.

- Beadle and Tatum concluded that each gene encodes for the structure of one enzyme. They called this relationship the one-gene-one enzyme hypothesis.

- The hypothesis was revised as not all proteins are enzyme. Some non-enzyme proteins are still gene products.

- It was later learnt many proteins are made up of more than one polypeptide. The one-gene-one polypeptide hypothesis is the final hypothesis.

DNA replication

- A specific replication scheme of DNA in a cell ensures that genetic information is passed down accurately to the next generation cells. This replication takes place before a cell starts to divide.

- Based on Watson and Crick’s double helix DNA model, three models of replication were proposed:1. The conservative model.2. The semiconservative model3. The dispersive model

- Matthew Meselson and Franklin Stahl devised a clever experiment that distinguished between the three models.

- Their experiment supported the semiconservative model of DNA replication.- E. coli is used in the experiment.- The hybrid DNA (14N15N DNA) exists in each generation.- In the conservative model, there will be no hybrid DNA formed. Therefore, this

model is not accepted.- In the dispersive model, the second generation will have no 14N14N DNA.

Therefore, this model too cannot be accepted.

DNA replication process

1. The DNA helicase enzyme unwinds the double stranded DNA to a replication fork ready for replication.

2. On the upper template strand, RNA primase attaches a short RNA primer in the 5’-3’ direction.

3. RNA primase is removed and free DNA nucleosides are added by DNA polymerase III to the RNA primer in the 5’-3’ direction.

4. This new DNA is called the leading strand because it is made in the same direction as the movement of replication fork.

5. On the bottom template stand, RNA primase synthesizes a shortr RNA primer in the 5’-3’ direction.

6. RNA primase is removed and DNA polymerase III adds DNA nucleosides to the RNA primer one after another in the 5’-3’ direction opposite to the replication fork to form a short length DNA.

7. This new short length DNA is called the lagging strand because it is made in the direction opposite to the movement of replication fork. The fragment produced is also called an Okazaki fragment.

8. The DNA unwinds some more and the leading strand is extended by DNA polymerase III by adding more DNA nucleosides. Thus, leading strand is synthesized continuously.

9. On the bottom antiparallel 3’-5’ template strand a new RNA primer is attached by DNA primase near the replication fork. This is followed by DNA polymerase III adding DNA nucleosides to the RNA primer one after another in the 5’-3’ direction opposite to the replication form to form another short length DNA.

10. Another short length DNA fragment is formed. This short length fragment is the second Okazaki fragment. Thus, the lagging strand is synthesized discontinuously.

11. The process repeats as the DNA continues to unwind. Since one new DNA strand (leading strand) is synthesized continuously and the other (lagging strand) is synthesized discontinuously, this model is also called the semidiscontinuous model for DNA replication.

12. The RNA primers are removed by a different type of enzyme called DNA polymerase I.

13. The Okazaki fragments are then sealed or joined up by DNA ligase to produce continuous chain.

14. The two new antiparallel continuous strands are formed.15. When replication is complete, two double-stranded daughter DNA molecules are

formed. Each has one parent strand and one daughter strand.

The genetic code

- The code on the DNA consists of three bases called the triplet genetic code.- Each triplet base is called a codon codes for one amino acid on the protein that

will be synthesized.- There are four different types of bases found in DNA: adenine, cytosine, thymine

and guanine.- Hence, there are 64 possible triplet codes.- Since there are 64 possible triplet codes to form 20 types of different amino acid,

more than one triplet code codes for one amino acid. Thus, the code is degenerate.- Out of the 64 codons possible, there are three codons, UAA, UAG and UGA that

do not have tRNAs with correspond anticodons.- These codons, called nonsense codons or stop codons serve as stop signals in the

mRNA. It marks the end of the translation for a polypeptide sequence.- AUG is the start codon which marks the beginning of the translation for a

polypeptide. It encodes the amino acid methionine.

Protein synthesis

- The instruction for making proteins comes from the gene.- Transcription is the synthesis of RNA under the direction of DNA. It occurs in the

nucleus. The stretch of DNA that is transcribed into RNA is called the transcription unit.

- There are 3 different types of RNA molecules directed by the DNA: mRNA, tRNA and rRNA.

- In translation, polypeptide is synthesized under the direction of mRNA. The cell translates the base sequence in the mRNA into the amino acid sequence of a polypeptides. This process occurs in the cytoplasm.

Transcription

- Transcription is divided into 3 stages:1. Initiation2. Elongation3. Termination

- The DNA sequence where RNA polymerase attaches and initiates transpcription is known as the promoter.

- The area that contains the RNA polymerase, DNA and the growing RNA is called the transcription bubbles.

RNA splicing

- The eukaryotic gene consists of exons and introns.- The exons code for amino acid whereas the introns do not.- Enzymes found in the eukaryotic nucleus cut the introns from the pre-mRNA.

This is called splicing.- The mature RNA the exits the nucleus and is translated in the cytoplasm.

Translation

- Before translation occurs, aminoacyl-tRNA molecules have to be synthesized.- Specific aminoacyl-tRNA synthetase enzymes bind tRNA to amino acids to form

aminoacyl-tRNA.- One aminoacyl-tRNA synthetase exits for each of the 20 amino acids used in

protein synthesis.

Translation is divided into initiation, elongation and termination.

Ribosome

Initiation

Elongation

Termination

Summary