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ENZYMES: KINETICS, INHIBITION, REGULATION. Kinetic properties of enzymes Study of the effect of substrate concentration on the rate of reaction.

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Presentation on theme: "ENZYMES: KINETICS, INHIBITION, REGULATION. Kinetic properties of enzymes Study of the effect of substrate concentration on the rate of reaction."— Presentation transcript:

1 ENZYMES: KINETICS, INHIBITION, REGULATION

2 Kinetic properties of enzymes Study of the effect of substrate concentration on the rate of reaction

3 - At a fixed enzyme concentration [E], the initial velocity Vo is almost linearly proportional to substrate concentration [S] when [S] is small but is nearly independent of [S] when [S] is large - Rate rises linearly as [S] increases and then levels off at high [S] (saturated) Rate of Catalysis

4 Leonor Michaelis and Maud Menten – first researchers who explained the shape of the rate curve (1913) During reaction enzyme molecules, E, and substrate molecules, S, combine in a reversible step to form an intermediate enzyme-substrate (ES) complex k 1, k -1, k 2, k -2 - rate constant - indicate the speed or efficiency of a reaction E + SESE + P k1k1 k2k2 k -1 k -2

5 The basic equation derived by Michaelis and Menten to explain enzyme-catalyzed reactions is V max [S] v o = K m + [S] The Michaelis-Menten Equation K m - Michaelis constant; V o – initial velocity caused by substrate concentration, [S]; V max – maximum velocity

6 Effect of enzyme concentration [E] on velocity (v) In fixed, saturating [S], the higher the concentration of enzyme, the greater the initial reaction rate This relationship will hold as long as there is enough substrate present

7 Enzyme inhibition In a tissue and cell different chemical agents (metabolites, substrate analogs, toxins, drugs, metal complexes etc) can inhibit the enzyme activity Inhibitor (I) binds to an enzyme and prevents the formation of ES complex or breakdown it to E + P

8 Reversible and irreversible inhibitors Reversible inhibitors – after combining with enzyme (EI complex is formed) can rapidly dissociate Enzyme is inactive only when bound to inhibitor EI complex is held together by weak, noncovalent interaction Three basic types of reversible inhibition: Competitive, Uncompetitive, Noncompetitive

9 Competitive inhibition Inhibitor has a structure similar to the substrate thus can bind to the same active site The enzyme cannot differentiate between the two compounds When inhibitor binds, prevents the substrate from binding Inhibitor can be released by increasing substrate concentration Reversible inhibition

10 Competitive inhibition Benzamidine competes with arginine for binding to trypsin Example of competitive inhibition

11 Binds to an enzyme site different from the active site Inhibitor and substrate can bind enzyme at the same time Cannot be overcome by increasing the substrate concentration Noncompetitive inhibition

12 Uncompetitive inhibition Uncompetitive inhibitors bind to ES not to free E This type of inhibition usually only occurs in multisubstrate reactions

13 Irreversible Enzyme Inhibition Irreversible inhibitors group-specific reagents substrate analogs suicide inhibitors very slow dissociation of EI complex Tightly bound through covalent or noncovalent interactions

14 Group-specific reagents –react with specific R groups of amino acids

15 Substrate analogs –structurally similar to the substrate for the enzyme -covalently modify active site residues

16 Inhibitor binds as a substrate and is initially processed by the normal catalytic mechanism It then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification Suicide because enzyme participates in its own irreversible inhibition Suicide inhibitors

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19 Regulation of enzyme activity Allosteric control Reversible covalent modification Isozymes (isoenzymes) Proteolytic activation Methods of regulation of enzyme activity

20 Allosteric enzymes have a second regulatory site (allosteric site) distinct from the active site Allosteric enzymes contain more than one polypeptide chain (have quaternary structure). Allosteric modulators bind noncovalently to allosteric site and regulate enzyme activity via conformational changes Allosteric enzymes

21 2 types of modulators (inhibitors or activators) Negative modulator (inhibitor) –binds to the allosteric site and inhibits the action of the enzyme –usually it is the end product of a biosynthetic pathway - end-product (feedback) inhibition Positive modulator (activator) –binds to the allosteric site and stimulates activity –usually it is the substrate of the reaction

22 PFK-1 catalyzes an early step in glycolysis Phosphoenol pyruvate (PEP), an intermediate near the end of the pathway is an allosteric inhibitor of PFK-1 Example of allosteric enzyme - phosphofructokinase-1 (PFK-1) PEP

23 Regulation of enzyme activity by covalent modification Covalent attachment of a molecule to an amino acid side chain of a protein can modify activity of enzyme

24 Phosphorylation reaction

25 Dephosphorylation reaction Usually phosphorylated enzymes are active, but there are exceptions (glycogen synthase) Enzymes taking part in phospho- rylation are called protein kinases Enzymes taking part in dephosphorylation are called phosphatases

26 Isoenzymes - multiple forms of an enzyme which differ in amino acid sequence but catalyze the same reaction Isoenzymes can differ in:  kinetics,  regulatory properties,  the form of coenzyme they prefer and  distribution in cell and tissues Isoenzymes are coded by different genes Isoenzymes (isozymes) Some metabolic processes are regulated by enzymes that exist in different molecular forms - isoenzymes

27 H 4 : highest affinity; best in aerobic environment M 4 : lowest affinity; best in anaerobic environment Isoenzymes are important for diagnosis of different diseases There are 5 Isozymes of LDG:  H 4 – heart  HM 3  H 2 M 2  H 3 M  M 4 – liver, muscle Lactate dehydrogenase – tetramer (four subunits) composed of two types of polypeptide chains, M and H Example: lactate dehydrogenase (LDG) Lactate + NAD + pyruvate + NADH + H +

28 Activation by proteolytic cleavage Many enzymes are synthesized as inactive precursors (zymogens) that are activated by proteolytic cleavage Proteolytic activation only occurs once in the life of an enzyme molecule Examples of specific proteolysis Digestive enzymes –Synthesized as zymogens in stomach and pancreas Blood clotting enzymes –Cascade of proteolytic activations Protein hormones –Proinsulin to insulin by removal of a peptide

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30 Multienzyme complexes - different enzymes that catalyze sequential reactions in the same pathway are bound together Multifunctional enzymes - different activities may be found on a single, multifunctional polypeptide chain Multienzyme Complexes and Multifunctional Enzymes

31 Metabolite channeling Metabolite channeling - “channeling” of reactants between active sites Occurs when the product of one reaction is transferred directly to the next active site without entering the bulk solvent Can greatly increase rate of a reactions Channeling is possible in multienzyme complexes and multifunctional enzymes


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