ENZYMES

Dr. KHUSHBU SONIAssistant Professor

AIMS,Dewas

o Enzymes are biological catalysts that speed up the rate of the biochemical reaction without undergoing permanent change in overall process.

o Substrate is reactant on which enzyme acts and converts it into a product.

o Zymogens (pro enzyme) are inactive form of enzymes. E.g. proelastase, chymotrypsinogen

Nomenclature of enzymesEnzymes are classified by two ways

Recommended name systematic name

o Trivial name (trypsin, pepsin)o Conveniento Easy to everyday useo Suffix –ase is used after substrate

e.g. lactase, ureaseo after type of reaction

e.g. oxidase

A systematic classification of enzymes has been developed by International union of biochemistry.

This classification is based on the type of reactions catalyzed by enzymes.

There are six major classes.

Enzyme Code (E.C.) = Four Digits

1. First (main class) = Type of Reaction 2. Second (subclass) = Type of Group involved 3. Third (sub-subclass) = denotes Substrate 4. Fourth = Individual enzyme name & serial numberE.C. 1. OxidoreductasesE.C. 2. TransferasesE.C. 3. HydrolasesE.C. 4. LyasesE.C. 5. IsomeraesE.C. 6. Ligases

1. OxidoreductasesCatalyzes a variety of oxidation-reduction reactionWith help of NADH, NADPH , FADH2 , FMNCommon examples

Dehydrogenases - Oxidases Peroxidases - Reductases

2. Transferases

Catalyzes transfer of functional group from one molecule to another.Carboxyl Methyl AcylGlycosyl amino

Kinase transfer of Phosphate group

3. Hydrolases

Cleavage of C-C, C-O, C-N & other Covalant bonds By addition of water.Example

Protease (Trypsin, Chymotrypsin, Pepsin ,Collagenase)

AmylaseLipasePhosphataseUrease

4. Lyases

Removal of group from substrates or break bonds by mechanism other than hydrolysis or oxidation.

ExampleAldolaseEnolaseFumaraseArginosuccinasePyruvate decarboxylaesHMG CoA lyase

5. Isomerases

produce Optical or Geometric isomer of substrateExample

RacemasesEpimerasesTriose phosphate isomeraseMutase

6. LigasesLink two substrate Usually with help of ATPExample

SynthetasePyruvate carboxylaseDNA Ligase

Co-factors and Co-enzymes

Some enzymes require molecules other than proteins for their action.

Apoenzyme = Enzyme (Protein moiety) = inactive

Holo-enzyme = Apoenzyme + Non-protein component

Non-protein component

Inorganic metal ion Organic molecule

Co-factor Co-enzyme

Metalloenzymese.g. Zn++ - carbonic anhydrase Mg++ - hexokinase

Prosthetic group

Co-substrate

Co-substrate

Prosthetic group

o When co-enzyme is loosely bound with enzyme

o After reaction, dissociates in altered state

o Need to be recycled by different reaction

o e.g. NAD+

o When co-enzyme is tightly bound with enzyme

o After reaction, returns to original form

o E.g. FAD

The term Apoenzyme is applicable to:

a) Simple enzyme

b) Protein part of conjugate enzyme

c) Organic co-factor of a conjugate enzyme

d) Inorganic co-factor of a conjugate enzyme

Zymogen is:

a) Enzyme modulator

b) Enzyme inhibitor

c) Enzyme precursor

d) Enzyme poison

Pyruvate carboxylase is:

a) Kinaseb) Lyasec) Transferased) ligase

Pyruvate decarboxylase is:

a) Oxido-reductaseb) Hydrolasec) Ligased) Lyase

Characteristics of enzymeso Most enzymes are three dimensional globular

proteins (tertiary and quaternary structure).

o Some special RNA species also act as enzymes and are called Ribozymes

o Water soluble

o Not consumed during reaction

o Their presence does not effect the nature and properties of end product.

o Enzymes are sensitive to change in pH, temperature and substrate concentration

o Active site: special pocket or cleft that binds substrates, co-factors and prosthetic groups and contains residue that helps to hold the substrate.

o generally occupy less than 5% of the total surface area of enzyme.

o has a specific shape due to tertiary structure of protein.

oContains substrate binding site and catalytic site.

oContains amino acid side chains involved in substrate binding and catalysis called as “catalytic residues”

oBinding occurs by non covalent forces.

oA change in the shape of protein affects the shape of active site and function of the enzyme.

Catalytic efficiencyenzyme catalyzed reactions are 1000 times faster than uncatlayzed one.

Turnover number defined as the number of substrate molecules transformed per second by one enzyme molecule.

Action of enzyme can be regulated depending on the production need of cell.

Cellular location: some localized in specific organelles, some are secreted out of the cell.

Specificity: ability of enzyme to discriminate between two competing substrates.

types

reactionsubstrate stereo

broad relative absolute

bond group e.g. urease e.g. hexokinase

e.g. alpha-amylasee.g. trypsin

L-lactate dehydrogenase

Lock and key model

o Proposed by EMIL FISCHER.

o Lock and key hypothesis assumes the active site of an enzymes are rigid in its shape.

o There is no change in the active site before and after a chemical reaction.

Koshland’s Induced Fit TheoryAccording to this theory, exposure of an enzyme to substrate cause a change in enzyme, which causes the active site to change it’s shape to allow enzyme and substrate to bind.

Reactions have an energy barrier

That energy barrier separate substrates and products.

It is difference between energy of the reactants and a high-energy intermediate that occurs during the formation of product.

Energy barrier = free energy of activation

Mode of action of enzymes

Rate of ReactionTo reach transition state

Substrate must contain sufficient energy.Enzyme

Rate of reaction is determined by the number of such energized molecules. In general, enzymes…

1. Lower the free energy of activation2. More molecules have sufficient energy to

pass3. Easily reach to transition state4. Faster the rate of the reaction.

Enzyme enhances rate of a biochemical reaction, as it:

a) Increases activation energy

b) Decreases activation energy

c) Increases substrate concentration

d) None of above

Mechanism of enzyme catalysis

Catalysis by

proximity

Metal ion catalysis

Covalent catalysis

Acid base catalysis

Catalysis can occur through proximity and orientation effects

o Enzymes are usually much bigger than their substrates

o By oriented binding and immobilization of the substrate, enzymes facilitate catalysis by:

1. bring substrates close to catalytic residues 2. Binding of substrate in proper orientation3. Stabilization of transition state by electrostatic interactions

Substrate stabilization in Transition stateThe active site acts as a flexible molecular template. Binds the substrate in a geometrically favorable manner.And activate transition state of the molecule By stabilizing the substrate in its transition state, the enzyme increases the concentration of the reactive intermediate.That can be converted to product.

Visualization of Transition state

Conversion of substrate to product can be visualized as being similar to removing a sweater from an uncooperative infant. We can en-vision a parent acting as an enzyme. Parent comes in contact with the baby (forming ES)Guide baby's arms to remove sweater. (ES transition state) Guidance (conformation) = facilitate the process.Removal of Sweater + Disrobed baby (Product)

o Enzymes contain catalytic residues at their active site

o Side chains of amino acids offer a variety of nucleophilic centers for catalysis

o Can form temporary covalent bond with

substrate molecule

o Enzyme-substrate intermediate

o At the end of reaction, the covalent bond must be broken to regenerate enzyme.

Covalent catalysis

Acid-base catalysis

o Active site may contains residue like histidine

o Participate in hydrogen ion transfer,

o by transferring hydrogen ion, the active site may:• Activate nucleophiles required in

catalysis

• Stabilize charged groups

• Facilitate electrostatic interactions that may stabilize transition state

Metal ion catalysis

o Metal ions like Zn, Mg, Fe etc.. are used as co-factor by various enzymes.

o Metal atoms lose electron easily and exist as cations

o The positive charges on metal ions allow them to:• Stabilize transient and intermediate

structures in the reaction• Assist in forming strong nucleophilic

group• Hold the substrate inside the active site

Carbonic anhydrase

Factors Affecting Enzyme Reaction

1. Substrate concentrationRate of reaction increases with substrate concentrationUntil Vmax is reached.At high conc. of substrate = enzyme full saturated with substrate.

2. TemperatureMaximum reaction velocity at Optimum

temperature.Optimum temperature for most human enzymes

is 35° - 40°C. Human enzymes start to denature above 40°C

temperature.

Effect of Temperature on Enzyme activity

3. pHo Concentration of H+ affects active site

o So Velocity reaction affected

o Change in pH can denature enzyme

o Optimum pH is different for different enzyme.

What change can occur at active site, because of change in pH?

Effect of pHIf the pH changes much from the

optimumChemical nature of the amino acids

can change. Change in Ionization of amino acid at

active site.Result in a change in the bonds. Active site will be disrupted.Enzyme will be denatured.

Different enzyme with it’s optimum pH

4. Enzyme concentration

Rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations.

5. Product concentrationAs product concentration increases,

enzymatic reaction slow down.

Higher Product concentration Inhibits reaction.

6. Enzyme activation Activation by co-factors.

In presence of certain metallic ions, some enzyme shows higher activity.

Salivary amylase = chlorideLipase = calcium

Conversion of an enzyme precursor. Specific proteolysis is a common method of

activating enzymes and other proteins in biological system.

Zymogen activation by proteolytic cleavage

Velocity &Vmax of reaction

Rate or Velocity of a reaction (V) is the number of substrate molecules converted to product per unit time.Vmax is the maximum velocity of the reaction.Expressed as µmol of product formed per minute.

Michaelis-Menten EquationReaction model

Leonor Michaelis and Maude MentenIn this model,

Enzyme reversibly combines with its substrate

Form an ES complexSubsequently yields productRegenerating the free enzyme.

where: S is the substrate E is the enzyme ES-is the enzyme substrate complex P is the product K1,K-1 and K2 are rate constants

Michaelis-Menten Equation

Km (Michaelis constant)It is the [S] for achieving half of the Vmax.

Km = Substrate concentration at ½Vmax.

Reflects the affinity of the enzyme for substrate.

Small KmHigh affinity of the enzyme for substrate.Because a low concentration of substrate

is needed to reach ½Vmax of velocity.

Large Km Low affinity of enzyme for substrate Because a high concentration of substrate

is needed to reach ½Vmax of velocity.

Assumptions in the Michaelis-Menten equation

Relative concentrations of E and S[S] is much greater than [E], so that the

percentage of total substrate bound by the enzyme at any one time is small.

Steady-state assumption [ES] does not change with time (the steady-

state assumption).The rate of formation of ES is equal to that of

the breakdown of ES (to E + S & to E + P).

Initial velocity

Initial reaction velocities (Vo) are used in the analysis of enzyme reactions. This means that the rate of the reaction is measured as soon as enzyme and substrate are mixed. At that time, the concentration of product is very small and, therefore, the rate of the back reaction from P to S can be ignored.

ENZYME REGULATION

Allosteric regulatio

nReversible covalent

modification

Proteolyticcleavage

Induction and

repression

o It permits changing needs of the cell to meet its energy and resource demands.

o If a product is available in excess, enzyme regulation could then divert the resources to other needy reactions.

Why?

o Regulatory enzymes : in a multi-step enzymatic process, there will be one enzyme which will be responsible for overall rate of that process.

o Key enzyme or rate limiting enzyme

o Can be affected by signal molecules

Allosteric regulationo Allosteric enzymes are a class of regulatory

enzymes.

o Large and composed of many subunits.

o Contains allosteric site different from active site.

o Regulatory molecules bind at allosteric site.

o Can be affected by regulatory molecules = allosteric effectors (modulator)

o Binding can enhance or reduce enzyme activity.

o Modulator may have positive effect or negative effect.

o Two types of allosteric enzyme based on nature of modulator:

Homotropic allosteric enzymesHeterotopic allosteric enzymes

o Typically, allosteric regulation occurs via FEEDBACK mechanisms.

oNegative feedback positive feedback

oAllosteric enzymes show variation in kinetics.

o They do not follow michaelis menten kinetics.

o They show sigmoidal curve instead of hyperbolic curve when velocity [v] is plotted against [s].

Positive feedback

Covalent modificationo Enzyme activity may be regulated by

reversible covalent modification.

o Separate enzymes are used to add or to remove modifying groups.

o Phosphorylation is the most common type.

o Addition of phosphate group to Ser,Tyr,Thr.

o ATP and GTP donates phosphate.

Zymogen activation by proteolytic cleavage

Induction and Repression Regulate the amount of enzyme.Act at Gene level.Altering rate of enzyme synthesis. Increase enzyme synthesis = InductionDecrease enzyme synthesis = Repression Induction / Repression = Slow (hours to days)Allosteric regulation = Fast (seconds to minutes)

Feedback regulation

ISOENZYMES

Catalyze the same reaction

Two or more polypeptide chains

Different polypeptide chains are products of different genes

Differ in AA sequence and physical properties

Separable on the basis of charge

Are tissue specific

“They are physical distinct forms of the same enzyme ”

Different allosteric effectors and different kinetics

Type of

LDH

Composition Location

LDH 1 HHHH MyocardiumLDH 2 HHHM RBCLDH 3 HHMM LungLDH 4 HMMM Kidney &

PancreasLDH 5 MMMM Skeletal

muscle & Liver

Creatine Kinase - DimerType of CK Compositio

nLocation

CK- 1 (CK-BB) BB BrainCK- 2 (CK-MB) MB MyocardiumCK- 3 (CK-MM) MM Skeletal

Muscle

Identification of Isoenzymes

1. Electrophoresis2. Heat stability : BALP3. Inhibitors4. Substrate specificity (Km value)o e.g. Hexokinase & Glucokinase

5. Cofactor requiremento e.g. Mitochondrial ICD – NAD+ dependent

Cytoplasmic ICD – NADP+ dependent6. Tissue location7. Specific antibody

Isoenzymes of Alkaline Phosphatase

Depending on number of sialic acid residue1. Alpha – 1 ALP (10%) Biliary Canaliculi2. Alpha – 2 heat labile ALP (25%) Hepatic cells3. Alpha – 2 heat stable ALP (1%) Regan Isoenzyme

Placental cell 4. Pre – beta ALP (50%) Bone disease5. Gamma – ALP (10%) Intestinal cells6. Leucocyte ALP Leucocyte

Organ Specific Enzyme

Heart CK-MB , AST (GOT) , LDH Liver ALT , AST , LDH , Alkaline

PhosphataseGamma Glutamyl Transferase

Pancreas

Lipase ,Amylase

Muscle Aldolase , CK-MM , CK-Total , AST

Bone Alkaline PhosphataseProstate Acid Phosphatase

(Prostate isoform – inhibited by Tartrate)

RBC LDH Acid Phosphatase (Erythrocyte isoform – inhibited by formaldehyde & cupric ion)

Principal Sources Diagnostically Important Enzyme

Liver Alanine aminotransferase(ALT)

Liver, Gall Bladder, Erythrocytes Skeletal muscle, Heart, Kidney ,

Aspartate aminotransferase(AST) I (cytosol) & II (mitochondria)

Hepatobilliary tract, Kidney Gamma Glutamyl Transferase

Hepatobilliary tract 5’ Nucleosidase Bone, Gall Bladder ,Liver, Intestinal mucosa, Placenta, Kidney

Alkaline Phosphatase (ALP)

Prostate, Erythrocytes Acid Phosphatase Pancreas ,Salivary glands, Ovaries

Amylase

Pancreas Lipase

Enzyme as Therapeutic Agents

1. Streptokinase & Urokinase• Lysis of intravascular clot• Use in myocardial infarction

2. Asparaginase• Used as anticancer drugs.

1. Glucose oxidase & Peroxidase (GOD-POD)

2. Urease3. ELISA test4. Restricted Endonuclease

Enzyme as Diagnostic Agents