Enzymes Dr Manjula Shantaram Ph D Biochemistry Dept

Enzymes Dr. Manjula Shantaram Ph. D Biochemistry Dept. , YMC, YU

Enzymes – Table of Contents Introduction Classification Mechanism of Action Part I Enzyme Kinetics Enzyme Inhibition Enzyme regulation Part II Isoenzymes Clinical Applications of Enzymes 05/74

Table of contents – Part I in detail • Introduction, definition Enzyme Vs Catalyst • Classification – IUB • Cofactor & Coenzyme • Metallo enzymes • Active site • Enzyme specificity • Mode(Mechanism) of Action of Enzymes Theories – Michaelis – Menton Theory Fisher’s Template Theory Koshland’s Induced fit Theory • Enzyme Kinetics Factors affecting Velocity – Km Value Significance 06/74

Background n Rate of a Reaction, Reversible & Irreversible Reaction and Reaction Equilibrium n Consider a chemical reaction, r 1 (k 1) n A+B C+D, where A & B are reactants, C & D are products, r 1 is the rate (velocity or speed) of the reaction and k 1 is the rate constant. n Since the rate of a reaction is directly proportional to the product of the concentrations of the reactants, n r 1 [A] [B]

Therefore, n r 1 = k 1 [A] [B], where k 1 is a rate constant. n Now consider a reversible reaction, r 1 (k 1) n A+B C+D r 2(k 2) n At the start, since there will be only the reactants and no products, the rate of forward reaction (left to right) will be maximum and that of backward reaction (right to left) is zero. n As the reaction proceeds, the concentrations of A and B decrease and those of C and D increase;

n and therefore, the rate of forward reaction decreases and that of backward reaction goes on increasing. n After sometime a stage will be reached when the rate of the forward reaction becomes equal to the rate of backward reaction. Then the system is said to have attained a state of equilibrium – chemical equilibrium. n Hence the concentrations of all the reactants and products at equilibrium will have become stationary. n That is, at equilibrium, r 1 = r 2 n Since r 1 = k 1 [A] [B] and r 2 = k 2 [C] [D], n At equilibrium, k 1 [A] [B] = k 2 [C] [D] [A] [B] = k 2 [C] [D] k 1

n [A] [B] = Keq (equilibrium constant) – law of n [C] [D] mass action, for reversible reactions n In a freely reversible reaction, the value of Keq is 1 and there is no energy change (ΔG = 0); n In an irreversible reaction the Keq value is very high (endergonic reaction; ΔG = +ve) or negligible(exergonic reaction; ΔG = –ve).

A Catalyst n For example: Consider hydrolysis of sucrose: n Sucrose + H 2 O H+ glucose + fructose. HCI n The role of HCl here is “ Catalyst” n Accelerates the rate of reaction many folds.

CATALYSIS n A catalyst increases the rate of a chemical reaction but remains unchanged chemically at the end of the reaction. The phenomenon is catalysis. Catalysts greatly enhance the rate of achievement of reaction equilibrium. n But they neither cause chemical reactions to take place nor change the equilibrium constant of chemical reactions. n Catalysts catalyze the forward and backward reactions equally. n A catalyst will only catalyze the reaction in thermodynamically allowed direction.

Activation Energy required for a reaction to take place at room temperature is the activation energy. n Catalysts accelerate chemical reactions by lowering the activation energy or energy barrier for a reaction to take place. n When a reactant acquires enough energy (activation energy) to undergo transition to form the product its energy status is said to be in transition state (T). n Thus, it can also be said that a catalyzed reaction needs less energy to move to transition state.

Mechanism n For a reaction to take place, reactant molecules must collide with each other with sufficient energy. n Thus each reaction is said to have an energy barrier. n Eg: Hydrolysis of Sucrose n Sucrose + H 2 O + 290 Kcal Glucose + Fructose (energy barrier) n The minimum amount of energy that the reactants must possess to overcome this barrier is called “activation energy”

q This Process is called “tunnelling through energy barrier” by providing alternate pathways. n Enzymes act by lowering the activation energy by the same mechanism. n Most of the reactions in the body have very high energy barriers, but enzymes accomplish these reactions at body temp, by lowering activation energy. Recall: n Eg: Sucrose + H 2 O+ 90 Kcal Sucrase Glucose + Fructose

Introduction Enzymes are defined as biological catalysts which are protein in nature. (Ribozyme is a biological catalyst made of RNA) Like all proteins, enzymes are synthesized by living cells; active both inside and outside the cell and even in cell free extract, colloidal in nature and heat labile. Compared to inorganic catalysts, enzymes are more specific, more efficient, larger in size and less stable. 07/74

General features of enzymes n There are millions of chemical reactions taking place in the body and all of them, except a few, are enzyme-catalyzed. n Very few reactions that are not enzyme catalyzed are called spontaneous or nonenzymatic reactions. n The reactant/s on which the enzymes act to catalyze the reaction are called the substrate/s of the enzyme. n Enzymes are much larger than the substrates they act on.

General features ……. . continued n Enzymes have active sites or active centers, where the catalysis takes place. n Active site is only a small portion of the enzyme. n The active site contains substrate binding site and catalytic site. n Enzymes are huge in size but with small active sites.

Clinical Importance of Enzymes n Enzymes play central role in health & Diseases (Eg: Inherited genetic diseases due to deficiency of enzyme). Any disease has an enzyme background. n Many drugs exert their action through enzymes. n Measurement of enzymes in blood is useful for diagnosis and follow up. n Therapeutic importance

Biological importance of enzymes n Enzymes catalyse multiple dynamic processes, which make life possible. Eg: Digestion & absorption, breakdown of food to give energy, muscle contraction, synthesis & maintenance of tissues, fighting against infection, etc. n (Commercial importance of enzymes is in industrial applications)

Nomenclature (Trivial Names) n Enzymes are named by adding suffix – ‘ase’ to the substrate n Ex: Lactase, sucrase, proteases n Nature of reaction n Ex: Dehydrogenase, transferase n Many enzymes are named after the reaction they catalyze.

Classification of enzymes n In 1964, IUB suggested a classification for enzymes which now widely accepted. n It is based on reaction type n As per this system enzymes are classified into 6 major classes, each of which is further sub divided into Sub class, Sub-sub class etc. n Each enzyme is given Enzyme – Code (EC) which is a 4 digit number. n Eg: Alcohol dehydrogenase EC is 1. 1

Enzyme Classification 1. Oxido-reductases - Redox reaction. Transfer of hydrogen or oxygen or electrons) 1. Alcohol Dehydrogenase Eg: CH 3 OH HCHO Alcohol Aldehyde NADH + 2. Lactate dehydrogenase Pyruvate Lactate NADH + H+ Oxido-reductases are further sub classified into oxidases, aerobic dehydrogenases, anerobic dehydrogenses, hydroperoxidases and oxygenases.

2. Transferases n Transfer of a group(other than hydrogen) from one substrate to another. 1. Glucose Hexokinase Glucose – 6 - Phosphate ATP ADP Alanine transaminase 2. Pyruvate Alanine Glutamate α Keto glutarate

3. Hydrolases n Hydrolysis(Splitting of an anhydride bond with addition of water, like ester, peptide glycosidic bonds). Eg: All GIT enzymes Lactose Lactase Glucose + Galactose Sucrose sucrase Glucose + Fructose

4. Lyases n Breaking of bond by other than hydrolysis. n Aldolase 1. Eg. Fructose -1, 6 Bis- Phosphate Glyceraldehyde -3 - n Phosphate DHAP n 2. Malate Fumarase Fumarate + Water

5. Isomerases Isomerization ( Intramolecular rearrangement of atoms) 1 Eg: Phosphohexose isomerase Glucose – 6 - Phosphate Fructose -6 -Phoshate 2. Eg: Phosphotriose isomerase Glyceraldhyde -3 - Phosphate DHAP Names of isomerases end with isomerases, epimerases, racemases or mutases.

6. Ligases n Joining two molecules by covalent bond at the expense of energy (ATP). They are synthetases. n ATP ADP + Pi n 1. Eg : Pyruvate + CO 2 Oxalo acetate Pyruvate carboxylase ATP ADP + Pi 2. Glutamic Acid + NH 3 Glutamine synthetase Pneumonic – OTHLIL (Oh Thank Heaven Learning Is Lively)

Cofactors n Non protein factors required for enzyme action. n These are small molecules, heat stable. They may be: 1) Organic molecules – called Coenzymes 2) Inorganic molecules – called activators, usually metal ions like Zn 2+ or Fe 2+, Cl- , Mg 2+ Without these cofactors, the enzyme does not exhibit any catalytic activity.

Cofactors……. continued Some enzymes also contain a non protein part which is tightly bound to the enzyme called prosthetic group. n Ex: Haem in Cytochromes n Protein part of the enzyme is called apoenzyme Apoenzyme + Cofactor or prosthetic group Holoenzyme

Coenzymes n Non protein organic cofactors required for enzyme action n Small molecules, dialysable, heat stable. n Many Coenzymes, like substrates, bind reversibly by non covalant bonds to the enzyme active site. n They undergo chemical change during reaction, regenerated & released at the end of the reaction. Hence they can be considered as co- substrates.

Coenzymes. . . continued Exception: Coenzymes FMN &FAD, Biotin, PLP are bound tightly to the enzyme by covalent bonds. n They are not specific to the enzyme (Enzyme has a specific coenzyme but a coenzyme can have many enzymes) (Ex: NAD has around 700 enzymes) n Hydrolases do not require coenzymes n Many coenzymes are vitamin derivatives. n Functions: Coenzymes act as carriers of various groups during the reaction (Addition or removal of a group from substrate to form the product):

Examples of Coenzymes Coenzyme NAD+, NADP+ FAD & FMN Coenzyme A Biotin Thiamine. Pyro phosphate(TPP) Tetrahydro folate(FH 4) Pyridoxal Phosphate(PLP) Methyl Cobalamine Derived from Niacin (Vit B 3) Riboflavin (Vit B 2) Pantothenic acid Biotin (B 7) Thiamine (Vit B 1) Group transferred Hydrogen/electron Folic acid One Carbon groups (methyl, formyl, etc) Amino/ Carboxyl or other Methyl Pyridoxine (Vit B 6) Cobalamine (Vit B 12) Acyl group CO 2 Aldehyde or Keto

Clinical Importance of coenzymes n Since many of the coenzymes are vitamin derived, deficiency of specific vitamin leads to specific coenzyme deficiency resulting in diseases. Eg: Pellegra, Megaloblastic anemia, etc. Inorganic Cofactors (Activators) These are metal ions like Zn, Mg, Fe. They are of 2 Types: n Metallo enzymes – Metal ions requiring enzymes where metal is bound tightly to enzyme. Eg: Carbonic anhydrase (Zinc). n Metal Activated enzymes- Metal is not tightly bound to the enzyme. Eg: Enolase & ATPase (Mg 2+) n Cl- (Non Metal) – Activator of salivary amylase

Active site of the enzyme n. Enzymes are big molecules compared to substrates. n. Active site of an enzyme is a small region of the enzyme where substrate binding & subsequent catalysis takes place.

Active Site

Salient features of Active Site: n Situated in a pocket or cleft of the enzyme molecule. n It contains substrate binding site & catalytic site. n Specific substrate binds at the Substrate binding site reversibly by non covalent forces. n Contains specific groups or atoms which facilitate the binding of correct substrate.

Salient features of active site. . . continued n. Catalytic Site is constituted by one or more amino acid residues called as catalytic residues which are involved in catalysis. ( Ser. , Thr etc are commonly found). n Cofactors required for some enzymes are also present as a part of the catalytic site.

Enzyme Specificity n Enzymes are highly specific to the reaction they catalyze. n Specificity of enzyme resides in active site because its shape is complementary to the substrate. n More specific when compared to an inorganic catalyst. n Types of Specificity 1. Absolute substrate specificity n 2. Broad substrate specificity

n Absolute substrate specificity: Here enzymes catalyze only one reaction n Glucokinase to form Glucose -6 – Phosphate n Urease to form Ammonia + CO 2 n L-Amino acid oxidase – does not act on Damino acids n Lactase to form glucose and galactose

Broad substrate specificity: Enzymes can catalyze more than one reaction Eg: Hexokinase acts on Hexoses (glucose, fructose, galactose). Proteases – trypsin, pepsin act on all proteins Aminopeptidases remove amino terminal end of all proteins.

Mechanism of enzyme action n The lowering of activation energy is explained by taking Michaelis –Menton model of enzyme catalyzed reaction. n As per this theory, enzyme(E) combines with substrate(S) to form enzyme – substrate complex(ES) which immediately dissociates to form product(P) releasing the free enzyme. E + S ES E + P (transition state)

Theories to explain formation of ES & subsequent catalysis: 1. Fischer’s template theory (Lock & Key model) 2. Koshland’s induced fit theory. Template theory • As per this theory, the active site of the enzyme is the template which is rigid & complimentary to the substrate. • It is just like correct key(substrate) which fits into the Lock(active site).

This theory explains the specificity but fails to explain flexibility shown by enzyme while binding substrate and during catalysis.

2. Koshland’s induced fit model n As per this theory, the active site is not rigid(flexible). The binding of substrate induces conformational changes in the active site. n It results in correct orientation of catalytic groups of active site with substrate leading to subsequent catalysis.

v. This is the most accepted model. v. Unlike the lock & key model, this model explains specificity & flexibility shown by enzyme molecules during substrate binding & catalysis.

ENZYME KINETICS n. Enzyme kinetics is the study of velocity/rate of an enzyme catalysed reaction n(Rate of change of substrate to product per unit time). n. Velocity is also referred to as enzyme activity.

Importance of Enzyme Kinetics 1. Understanding the mechanism of enzyme action & inhibition. 2. Understanding disease processes. 3. Help in clinical diagnosis by measuring certain enzymes in disease conditions. n International unit of enzyme activity (U or U/liter) for clinical enzyme assays is the micromoles of substrate converted to product per minute in a liter of body fluid (e. g. blood).

Factors affecting enzyme activity 1) Enzyme concentration 2) Substrate concentration 3) Temperature 4) p. H Other factors include presence of coenzymes, activators or inhibitors, concentration of product, etc. .

1. Effect of enzyme concentration n The velocity ( i. e. Initial velocity or Vi )of an enzyme catalysed reaction increases when enzyme concentration is increased keeping all other factors constant. n Vi is directly proportional to the enzyme concentration. n A straight line is obtained by plotting Vi against enzyme.

This property of enzyme is made use in determining the activity of serum enzymes for the diagnosis of diseases. Velocity Vi 0 Enzyme concentration

2. Effect of temperature n The velocity (vi) of an enzyme-catalyzed reaction increases when temperature of the medium is increased, reaches a maximum and then falls. n A bell-shaped curve is usually observed when velocity is plotted vs. temperature. n The temperature at which the velocity is maximum is called the optimum temperature.

Velocity

Explanation for Effect of Temperature: n Increase of temperature leads to increase of kinetic energy of both substrate and enzyme molecules, which in turn leads to increase in the strength and frequency of collision between substrate and enzyme molecules resulting in the increase of enzyme activity. n However, when temperature is very high (more than 55 o. C), heat denaturation and consequent loss of tertiary structure of proteins occur. Hence the activity of the enzyme decreases.

Effect of temperature …. contd. n The optimum temperature of most enzymes is between 40 to 50 o. C. n Most human enzymes are stable up to 55 o. C. n Enzymes present in Thermus acquaticus bacteria living in hot spring, are stable and active even in boiling water. (This has application in polymerase chain reaction[PCR] – a DNA analysis technique).

3. Effect of p. H n Each enzyme has an optimum p. H on both sides of which the velocity (i. e. initial velocity or Vi) will be drastically reduced. n The graph will show a typical bellshaped curve when velocity is plotted vs. p. H.

Explanation for effect of p. H n Alteration in p. H will lead to alteration in the charged state of enzyme or substrate (or both). This may change binding of substrate to enzyme or catalytic activity of the enzyme itself. n At extreme p. Hs (at strong acidic and alkaline conditions) enzyme molecule gets denatured and therefore its activity is drastically reduced. n Usually enzymes have the optimum p. H between 5 and 9. [Some important exceptions are pepsin (12), alkaline phosphatase (9 -10) and acid phosphatase (4 -5)]

4. Effect of substrate concentration n Since concentration of enzyme is far less than that of substrate molecules, the interaction of enzyme with substrate obeys what is called as saturation kinetics. n The velocity depends solely upon the extent of saturation of enzyme molecules with substrate (vi [ES]). n In the initial phases; as substrate concentration is increased, velocity also increases correspondingly, but in the later phase the curve flattens, i. e. velocity will not increase as the substrate concentration is increased.

½Vmax

Effect of substrate concentration III Phase

n This can be considered under 3 phases n 1 st phase: - is a straight line n At low [ S] , velocity is directly proportional to [ S]. Ie, as the [ S] increases, Velocity also increases n So, it gives a straight line. It is a first order kinetics. n II Phase : is a Curve. n As the [S] is increased, velocity is increased, but it is not proportionate. So, it gives a curve. It is in between first and zero order kinetics.

III Phase: Is a plateau When [S] is increasing, slowly enzymes are getting saturated, since concentration of enzyme is limited. So, velocity slowly starts declining. When [S] is increased still further, at a particular point, all the enzymes are fully saturated & maximally working. There are no free enzymes to carry out the reaction. That is why the velocity reaches maximum & becomes independent of [S]. It is a zero order kinetics. We get a rectangular hyperbolic curve.

Michaelis – Menten Equation & Km Value Two scientists Leonor Michaelis & Maud Menten, deduced an equation which relates the velocity of an enzyme catalysed reaction with substrate concentration.

Michaelis – Menten Equation & Km Value: n The equation is as follows: Vmax [S] Vi = Km + [S] Where, n Vi = Initial Velocity n V max = Maximum Velocity n [S] = Substrate concentration n Km = Michaelis Constant n Km & V max are the two constants for an enzyme.

n When Vi = ½ Vmax n Km = [ S] n In an enzyme catalysed reaction, K 1 K 2 n E + S ES E + P K 3 n It was also shown that n Km = K 2+K 3 K 1 n (In Km, K stands for a constant and m for Michaelis) n [This equation is for a rectangular hyperbole (y = ax/ b+x)]

n Km & its significance: n Definition: Km is the substrate concentration required to attain ½ Vmax. n i. e , Km = [S] When Vi = ½ Vmax n - It denotes that 50% enzyme molecules are saturated with substrate molecules at that particular substrate concentration. n - Expressed as moles/ liter n ( It ranges from 10 -5 – 10 -2 moles /liter) n Calculation of Km Value can be calculated using Michaelis Menten plot, by dropping a perpendicular to x axis at ½ Vmax.

Significance of Km Value n Km is the characteristic feature of an enzyme. It is constant for an enzyme. Aptly called as signature of an enzyme. n Km is the measure of the affinity of enzyme for its substrate. Low Km High affinity for substrate High Km Low affinity for substrate

. Ex: Glucokinase & Hexokinase catalyze the same reaction Glucose Glucose 6 - Phosphate Km value for Glucokinase = 10 m mol/Lt Km value for Hexokinase = 0. 05 m mol/Lt Therefore, Hexokinase has more affinity for glucose

n By knowing Km Value , one can know the natural substrate of an enzyme having more than 1 substrate. One having least Km is the natural substrate. Eg: Hexokinase can phosphorylate glucose, fructose & galactose. But Km value of hexokinase with glucose is the least which signifies that glucose is the natural (preferred ) substrate for hexokinase. n Determination of Km helps in study of mechanism of enzyme inhibition( Refer enzyme inhibition).

n. Km values of isoenzymes are different for the same substrate(Refer Isoenzymes). n. Enzyme assays in clinical labs & research are based on the knowledge of Km.

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