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Enzymes

ENZYMES Enzymes are highly specialised proteins that act as catalysts in biological systems (biocatalysts). Enzymes are highly specific for their substrates

NOMENCLATURE AND CLASSIFICATION OF ENZYMES International Union of Biochemistry (IUB) devised a new system of nomenclature in 1964. A four-digit Enzyme Commission (E.C.) number is assigned to each enzyme. The first digit represents the class, the second digit indicates the subclass, the third digit represents the sub-subclass and the fourth digit specifies the individual enzyme. Enzymes are classified into six major classes as per the IUB system of enzyme classification.

CLASIFICATION OF ENZYMES (Continued…

CLASIFICATION OF ENZYMES …Continues) (Continued…

CLASIFICATION OF ENZYMES …Continues) (Continued…

…Continues)

ENZYME STRUCTURE Enzymes are proteins Apoenzyme Coenzyme Holoenzyme

ACTIVE SITE The small cleft-like portion of an enzyme where the substrate(s) binds and catalysis occurs is known as the active site or active centre. Substrate binding site Catalytic site A diagrammatic representation of an enzyme and its active site

COENZYMES Coenzymes of B-complex vitamins

Coenzymes which are not related to vitamins

ENZYME SPECIFICITY The active site in a specified conformation on an intact enzyme molecule is largely responsible for the enzyme specificity. There are three types of specificity: 1. Stereo specificity 2. Substrate specificity 3. Reaction specificity

HOW DO ENZYMES WORK? Transition state Ground state Activation energy Enzymes act by reducing the activation energies Binding energy

Effect of enzyme on activation energy in a reaction (S =substrate; P = product).

To summarise, the energy required to surmount the activation barrier is the activation energy. Enzymes catalyse the reactions by lowering the activation barrier. The binding energy resulting from weak non-covalent interactions between the substrate and the enzyme reduces the activation energy. Enzymes do not alter the reaction equilibria, they only enhance the reaction rates by lowering activation energies.

MECHANISM OF ENZYME ACTION 1. Lock and key model 2. Induced fit theory 3. Substrate strain theory

Different models explaining enzyme–substrate (ES) complex formation (A) Lock and key model, (B) Induced fit theory, (C) Substrate strain theory.

MECHANISM OF CATALYSIS Acid-base catalysis Covalent catalysis Metal ion catalysis

FACTORS AFFECTING ENZYME ACTIVITY The various factors that influence enzyme activity are concentration of enzyme concentration of substrate concentration of product Temperature pH.

EFFECT OF CONCENTRATION OF SUBSTRATE ON ENZYME VELOCITY The rate of an enzyme catalysed reaction increases as the substrate concentration increases until it reaches a maximal rate, which remains constant despite further increases in substrate concentration. The substrate concentration (expressed in moles/L) required to produce half-maximum velocity (1/2 Vmax) is known as Km or Michaelis–Menten constant.

Effect of substrate concentration on enzyme velocity.

Km or Michaelis–Menten constant can be derived as follows: The relationship between substrate concentration (S) and enzyme velocity (V) can be shown as follows: (Continued…

When the measured velocity (V) is exactly one-half Vmax …Continues) When the measured velocity (V) is exactly one-half Vmax In Km, K stands for a constant and m stands for Michaelis and Menten.

EFFECT OF TEMPERATURE ON ENZYME VELOCITY

EFFECT OF pH ON ENZYME VELOCITY

ENZYME INHIBITION Enzyme inhibition can be broadly classified into three categories. 1. Competitive inhibition 2. Non-competitive inhibition 3. Allosteric inhibition

COMPETITIVE INHIBITION The inhibitor binds with the enzyme through non-covalent interactions and hence the pro cess is readily reversible. In competitive inhibition, the Km value increases whereas Vmax is unchanged.

Velocity versus substrate plot in competitive inhibition

DRUGS ACTING AS COMPETITIVE INHIBITORS

NON-COMPETITIVE INHIBITION The inhibitor binds to the enzyme covalently and inactivates it making the process essentially irreversible. In non-competitive inhibition, the Km value is unchanged while Vmax is lowered.

Velocity versus substrate plot in non-competitive inhibition

REGULATION OF ENZYME ACTIVITY 1. Allosteric regulation 2. Covalent modulation 3. Proteolytic trimming 4. Compartmentation 5. Control of enzyme synthesis and degradation

ALLOSTERIC REGULATION Allosteric enzymes Allosteric modulators Cooperativity of allosteric enzymes Classification of allosteric enzymes K class allosteric enzymes V class allosteric enzymes Feedback regulation

Effect of substrate concentration on allosteric enzymes.

Examples of allosteric enzymes

COVALENT MODULATION Regulatory enzymes or rate-limiting enzymes Reversible protein phosphorylation Protein kinases Protein phosphatases

Regulatory enzymes (rate-limiting enzymes) controlled by covalent modulation

ISOENZYMES The multiple molecular forms of an enzyme catalysing the same reaction are called isoenzymes. They differ in their physical and chemical properties such as structure, Km, Vmax, pH, electrophoretic mobility and their susceptibility to inhibitors. Isoenzyme variants are coded by different genes. Allelozymes Hybrid isoenzymes Isoforms

CREATINE KINASE (CK) Normal serum levels of CK are 15–100 U/L in males and 10–80 U/L in females. Lohmann’s reaction In myocardial infarction, CK levels (CK–MB isoenzyme) start to rise within 3–6 hours of infarction. Isoenzymes of CK

LACTATE DEHYDROGENASE (LDH) Normal levels of LDH in serum are 100–200 U/L Isoenzymes of LDH

ALKALINE PHOSPHATASE (ALP) The normal serum levels of ALP range between 40 and 125 U/L. Increased ALP levels are most commonly associated with bone disease and hepatobiliary disease (Continued…

…Continues) Isoenzymes of ALP

Plasma specific enzymes Plasma non-specific enzymes Units of enzyme activity

DIAGNOSTIC MARKERS OF MYOCARDIAL INFARCTION (MI)

ENZYMES USED AS THERAPEUTIC AGENTS

ENZYMES USED IN LABORATORY MEASUREMENTS AND GENE TRANSFER