DR. S.CHAKRAVARTY MD

Enzymes

Learning objectives

Define and classify enzymes based on the IUPAC agreement. Give examples to each class

Classify cofactors and give examples for various types

Discuss the general properties of enzymes and list the important functions

Define KM, Vmax, Tranistion state and activation energy of enzymes

Discusss the factors affecting enzyme activity

Explain the Michaelis Menten reaction and differentiate it from lineweaver burk plot. Classify various types of enzyme inhibitions

Definition

Thermolabile biocatalysts which enhances a chemical reaction without undergoing any chemical change.

Properties :1. Specific for a reaction2. Does not dictate the direction of a reaction3. Increases the rate of a reaction by several

thousand times4. Are proteins – except ribozymes (RNA)5. Lowers the activation energy of a reaction

Enzyme terminology

Simple enzyme – made only of proteins

Complex enzyme – also called Holoenzyme 1. Apoenzyme – protein part2. Non-protein part -

Prosthetic group –with covalent interaction ( usually metals)

co-enzyme – without covalent interaction (vitamins )

Co-enzymes

Group I: take part in reactions transferring hydrogen atoms or electrons

VITAMIN COENZYME FUNCTION

Riboflavin FMNFAD

Redoxreactions

Niacin NADNADP+

Redoxreactions

Co-enzymes

Group II: take part in reactions transferring groups other than hydrogen

Vitamin Coenzyme Group transferred

Function

Thiamine TPP Hydroxy ethyl Transketolase,oxidative decarboxylation

Pyridoxine PLP Amino or keto Transamination reaction

Folic acid FH4 One carbon group

One carbon metabolism

Biotin Biotin Carbon dioxide Carboxylation reaction

Enzymes with metals

Metalloenzyme – metal is the indegenous part of the enzyme itself. Seperation causes disruption

Metal activated enzymes – required for

activation but not indegenous to the enzyme.

Metalloenzymes

Metal Enzyme containing the metalZinc Carbonic anhydrase, Alcohol dehydrogenase,

Iron Catalase, Peroxidase, Cytochrome oxidaseXanthine oxidase

Magnesium Hexokinase, Enolase,Glucose-6-phosphatase Phospho fructo kinase

Manganese Enolase, hexokinase

Copper Tyrosinase, Lysyl oxidase

Classification of enzymes: based on function

1. Oxidoreductases

2. Transferases

3. Hydrolases

4. Lyases

5. Isomerases

6. Ligases

c. Hydroperoxidase – use hydrogen peroxide as substrate to form water

Ex: catalase, peroxidase

2H2O2 ---------- 2H2O +O2

d. Oxygenases - Monooxygenase – single atom of oxygen (hydroxylase) Ex: Phenylalanine hydroxylase

A-H+O2+ZH2 ---------- A-OH +H2O +Z

Dioxygenase – incorporate both oxygen atoms Ex: Homogentisic acid dioxygenase

A+O2 --------- AO2

2.Transferases

Transfer of groups other than hydrogen:

a. Aminotransferases : transfer of amino groups Ex: Aspartate transaminase , Alanine transaminase

b. Acyl transferases : transfer of acyl groups which requires co-enzyme A

Ex: choline acetylase

C. Methyl transferase: transfer of methyl groups Ex: Homocysteine methyl transferase

D. Phosphotransferase (kinases) – transfer of phosphate groups. Ex : Hexokinse

3.Hydrolases

Clevage of the substrates by addition of water:A. Hydrolysis of carbohydrates – maltase,

lactase, sucrase etc.

B. Hydrolysis of triglycerides – lipase

C. Hydrolysis of proteins – pepsin, trypsin, etc

D. Phosphatases – phosphodiesterase and glucose -6-phosphatase.

4.Lyases

Cleavage of substartes or removal of groups by mechanism other than addition of water.

A. Decarboxylases – removal of carbon dioxide from substrates (require B6)

Ex : histidine decarboxylase

B. Phosphorylases – clevage of substrates by addition of phosphoric acid

Ex : Glycogen phosphorylase

5.Isomerases

Catalyze intramolecular rearrangement, catalyze conversions between optical, positional and geometric isomers.

A. Aldose ketose isomerase B. Epimerase C. Racemase – interconversion of D and L formsD. Mutase – transfer of chemical group from

one position to another in th same molecule. Ex: glucose -6-po4 -- glucose-1-po4

6.Ligases

Condensation of two molecules to form one molecule using energy in the form of ATP.

A. DNA ligase

B. Synthetase/synthase Glutamic acid +ammonia -------------- Glutamine

Glutamine synthase

ATP

Catalysis occurs at the active site

Features of active site

Occupies only a small portion of the whole enzyme

Situated in a crevice or cleft of the enzyme

Possesses a substrate binding site & a Catalytic site

The substrate binds at the active site by weak noncovalent bonds

The amino acids usually found at the active site are serine, lysine, histidine, arginine, cysteine

Theories explaining the binding of substrate to the enzyme

Fischer’s template theory (lock and key model of enzyme attachment :

According to this theory the active site of the enzyme is rigid. only a specific substrate complementary to the active site fits to it just as a key fits to its proper lock

Koshland’s induced fit theory:

According to this theory the active site is not rigid & pre- shaped.

The interaction of the substrate with the enzyme induces a conformational change at the active site so that proper alignment of the catalytic residues occur & the substrate fits in the active site

E P+

+

ES

ComplexS+ E

Koshland’s Induced Fit Theory

Fischer’s template theory

E S

E S

E S+ ES

Complex E P

+

+

Mechanism of action

Lowering of activation energy

Activation energy is defined as the energy required to convert all molecules of a reacting substance from ground state to transition state

Higher the activation energy ,slower the reaction & vice versa

Activation energy

Reactant [ground state]

Product[Transition state]

A BA*

Activation energy (Ea)

Mechanism of action

Mechanism of enzyme activity

1. Catalysis by proximity

2. Acid-base catalysis

3. Catalysis by strain

4. Covalent catalysis

Catalysis by proximity

High substrate concentration –more frequent encounter and greater will be the rate.

Enzymes bind substrate at active site high local substrate concentration

Orientation of molecules for better bonding

Acid-base catalysis

The amino acids at the active site contribute to catalysis by acting as acids or bases

Histidine is often the residue involved in these acid/base reactions, since it has a pKa close to neutral pH and can therefore both accept and donate protons.

Catalysis by strain

Enzymes bind to their substrates in a conformation slightly unfavorable for the bond present in the molecule which will undergo clevage.

Resulting strain stretches the bond and breaks it.

Covalent catalysis

Involves formation of a covalent bond between the enzyme and one or more substrates

Modified enzyme becomes a reactant

Creates new reaction pathway whose activation energy is lower

Serine, histidine, cysteine are involved.

Enzyme kinetics

Quantitative measurement of rates of enzyme catalyzed reaction and the study of factors that affect the rate of reaction.

A + B P + Q

The term substrate and products in the above are arbitrary in a reaction because the reaction can take place in both directions.

The direction of the reaction is dictated by the thermodynamics (change in free energy or delta G) of the reaction to which it favours.

Bioenergetics

Also called biochemical thermodynamics

Study of the energy changes accompanying chemical reactions

Describes the transfer and utilization of energy in biologic systems

Laws of Thermodynamics:

First law of thermodynamics: The total energy of a system, including its surroundings, remains constant

Derivation:

H = Q – WEnthalpy Heat Workor Heat content absorbed done

Second Law of Thermodynamics

The total entropy of a system must increase if a process is to occur spontaneously

Entropy:

# Extent of disorder or randomness of the System

# Maximum in a system as it approaches true equilibrium

Q = T Δ S Heat Temp Entropy

Direction and equilibrium of a reaction

If G = negative i.e., free energy of product is less than free energy

of substrates, the direction of the reaction is from left to right.

these reactions are said to be spontaneous

If G = positive i.e., free energy of the product is greater than the

free energy of the substrates, the direction of the reaction is from right to left favoring substrate formation.

If G =0, then the reaction is in equilibrium.

USMLE !

Delta G is negative Delta G is positive

Actual free energy change

Spontaneous and Exergonic Non-spontaneous and Endergonic

Δ G of two consecutive reactions are additive .

As long as the sum of the Δ Gs of the individual reactions is negative, the pathway can potentially proceed to completion even if some of the individual component reactions of the pathway have a positive Δ G .

Glucose + Pi glucose 6-P + H2O ΔG° = +3.3. kcal/mol

ATP + H2O ADP + Pi ΔG° = -7.3 kcal/mol

Glucose + ATP Glucose 6-P + ADP ΔG° = -4.0 kcal/mol

Exergonic and Endergonic reactions -Coupled USMLE !

Endergonic process proceed by coupling to exergonic process

The conversion of metabolite A to metabolite B occurs with release of free energy

• Free energy is required to convert metabolite C to metabolite D

An endergonic process cannot exist independently but must be a component of a coupled exergonic-endergonic system where the overall net change is exergonic.

Factors affecting the rate of enzyme catalyzed reaction

1. Enzyme concentration

2. Substrate concentration

3. Temperature

4. pH

5. Product concentration

6. Presence of activators & Inhibitors

Enzyme concentration

1. Effect of enzyme concentration [E] on velocity of reaction [v]

As the substrate concentration is increased the velocity also correspondingly increased in the initial phases but the curve flattens afterwards.

•A rectangular hyperbola is obtained

2. Substrate concentration

Substrate concentration

½ Vmax

Km

Vmax

First order reaction

At low substrate concentration the velocity of the reaction is directly proportional to the substrate concentration. This is first order reaction

Zero order reactions

At high concentration the velocity of the reaction is independent of substrate concentration. This is zero order reaction

Substrate concentration on the reaction rate :

3. Effect of temperature

The velocity of enzyme reaction increases when the temperature is increased reaches a maximum & then falls

The temperature at which maximum amount of substrate is converted to the product per unit time is called the optimum temperature

Effect of temperature

Increase in velocity of the reaction

Increase in temperature results in high activation energy of the molecules & more molecular collision & interaction for the reaction to proceed faster

Decrease in velocity of the reaction

When the temperature is increased more than the optimum temperature Denaturation of the enzyme occurs

4. Effect of pH

The pH decides the charge on the amino acid residues at the active site. The net charge on the enzyme would influence the substrate binding & catalytic activity

The accumulation of reaction products generally decreases the enzyme velocity.

The products combine with the active site of the enzyme & form a loose complex & thus inhibit the enzyme activity.

5. Effect of product concentration

Activators: Substances that increases the enzyme activity.

Eg : Zinc activates carbonic anhydrase NAD+ activates LDH

Inhibitors: substances that decreases the enzyme activity

Eg : Fluoride inhibits Enolase.

Cyanide inhibits cytochrome oxidase.

6.Factors affecting enzyme activity

Specificity of enzyme activity

Sterospecificity Reaction specificity Substarte specificity – Absolute Relative Broad

The same substrate can undergo different type of reaction. Each reaction is catalysed by a separate enzyme

Pyruvate Acetyl coAPDH

LactateLDH

Oxalo acetate

Carboxylas

eTransaminase

Alanine

Malate

Mal

ic e

nzym

e

Reaction specificity

Substrate specificity

Absolute specificity : Enzymes that can act only on one substrate & can catalyse only one reaction

Eg : Glucose Glucose-6-phosphate

Group specificity : Carboxy peptidase & amino peptidase are exopeptidase which hydrolyse peptide bond in the vicinity of free COOH or NH2 groups respectively

Broad specificity : Hexokinase acts on Glucose, Fructose, Mannose etc

Glucokinase

Michaelis –menten graph

X- axis – substrate concentration

Y-axis – velocity of the reaction (V)

Vmax = maximum velocity a reaction can reach. Directly proportional to amount of the enzyme and substrate

Km = substrate conc at the half maximal velocity.

Enzyme concentration is kept constant. As the substrate concentration increases the velocity increases but after some conc.. The graph flattens due to enzyme saturation. The highest point on the graph is called Vmax.

Representation of enzymes at various substrate concentration

Michaelis menten equation

Vi =Vmax [S]

Km + [S]

1) When substrate conc < Km value (point A), Vi will be directly proportional to substrate conc

2) When substrate conc = Km value(point B), vi wil be half the maximal velocity

3) When substrate conc > Km value(point C), Vi wil be equal to Vmax and independent of substarte conc on further increases.

What does the Km value say about a reaction?

High Km value means – the enzyme has low affinity for the substrate .

We have to load a large amount of substrate for the reaction to attain Velocity.

Low Km value means - the enzyme has very high affinity for substrate.

Even if small amounts of substrates are there the reaction will attain velocity

Affinity / binding is inversely proportional to Km value.

Binding is good when shapes of both enzyme and substrate match

Hill’s equation;

Y=ax+b

Conversion of michaelis mentan equation which is hyperbolic to hill’s equation which is linear.

Lineweaver –Burk Double reciprocal plot

X axis = 1/S

Point of intersection on x axis = (-1/Km) value

Y axis = 1/V

Point of intersection on Y-axis(slope) = 1/Vmax

Inverse plot of michaelis menten – both S conc and velocity plotted by taking 1/S and 1/V on x and y axis. This was done to make the equation linear.

Enzyme inhibition

A substance that can inhibit enzyme activity is called enzyme inhibitor:

Types : 1. Competitive inhibition2. Non-competitive inhibition3. Uncompetitive inhibition4. Mixed inhibition

Competitive inhibition

So in competitive inhibition:

Km is increased Vmax remains

unaltered because the number of enzymes remain the same.

Inhibitor resembles substrate in shape.

Substrate competes with catalytic site.

Inhibition is reversible because the inhibitor forms non-covalent bonds

Inhibition can be reversed by increasing substrate conc..

Competitive inhibition:

Lineweaver plot:

Michaelis menten plot :

Methanol Poisoning

“Wood” alcohol and antifreeze contain high methanol concentrations

Methanol poisoning causes decreased blood pressure and body temperature, and an increase in respiratory rate

Methanol + NAD+ Formaldehyde + NADH + H+

Ethanol + NAD+ Acetaldehyde + NADH + H+

Alcohol dehydrogenase has a slightly lower Km for ethanol compared to methanol

The additional benefit of the ethanol reaction is that acetaldehyde is less physiologically damaging than formaldehyde, which is toxic to the retina and can cause permanent blindness if doses are high for prolonged periods of time

Displaced methanol can be safely excreted by the kidneys

Alcohol Dehydrogenase

Alcohol Dehydrogenase

Methotrexate Structural analogue of folate inhibits folate reductase

Dicoumarol Structurally similar to Vit Kanticoagulant

Sulfonamide antibiotics structural analogues of PABA inhibits folate synthesis in bacteria Non-toxic to humans humans don’t synthesize PABA

Non-competitive inhibition:

Inhibitors bind to site other than substrate binding site.

Covalent bond formation – occurs

Loss of enzyme activity.

So Vmax decrease but Km unaltered.

Non-competitive inhibition

Michaelis menten plot: Lineweaver plot:

Uncompetitive inhibition

Inhibitor binds to the enzyme substrate complex.

Increased Substrate affinity apparently decreases the Km but Vmax is decreased because the enzyme will take a long time to separate from the complex.

Lineweaver plot:

Km value decreases

Vmax also decreases

Uncompetitive inhibition

Summary of inhibition:

Enzyme inhibition Km Vmax

Competitive inhibition

Increases Unaltered

Non competitive inhibition

Unaltered Decreases

Uncompetitive inhibition

Decreases Decreases

Mixed Increases Decreases

Enzyme regulation

The process by which cells can turn on, turn off, modulate the activities of various metabolic pathways

For coordinating various processes as per the physiological needs of the body.

Every pathway has enzymes which are the rate limiting/ or key enzymes

Types of enzyme regulation

Coarse regulation – Regulating enzyme activity

Fine regulation – Regulating enzyme concentration.

Coarse Regulation:

Covalent modification

Allosteric regulation

Feed back inhibition

Compartmentalization

Fine Regulation:InductionRepression

Types of enzyme regulation

Allosteric Regulation

These are the enzymes having one “Catalytic site” & another separate site called “Allosteric site” where the modifier binds & regulate the enzyme activity.

Catalytic site (substrate )

Allosteric site (allosteric compound)

Enzyme

Substrate

Allosteric activator

Enzyme Product

+

Enzyme substrate

Complex

Allosteric Activation

Enzyme

Substrate

Allosteric inhibitor

No enzyme substrate

Complex

Allosteric inhibition

Sigmoid curve

Positive modifier

Substrate concentration

Negative modifier

Allosteric regulation

• Addition of a group to the enzyme by a covalent bond or removal of a group by cleaving a covalent bond

Types

Covalent modification

Adenylation - deadenylation

Ribosylation

Methylation

Acetylation

Phosphorylation-dephosphorylation

Feedback inhibition

The process of inhibiting the first step by the final product, in a series of enzyme catalysed reactions of a metabolic pathway

A B C D Ee1 e2 e3 e4

E (-)

Compartmentalization

Localisation of enzyme

Metabolic pathway

Cytoplasm Glycolysis, fatty acid synthesis

Mitochondria Krebs cycle, ETC, Fatty acid oxidation

Peroxisomes Long chain fatty acid oxidation

Both mitochondria & cytosol

Urea cycle, Heme biosynthesis