Enzymes kinetics

ENZYME KINETICS AND FACTORS AFFECTING IT

1. Pt. RAVISHANKAR SHUKLA UNIVERSITY, Raipur S.o.S in Biotechnology TOPIC - ENZYME KINETICS GUIDED BY : Dr. Kamlesh K Shukla SUBMITTED BY – P . Sujata M.Sc. 2 nd Sem

2. Content Enzyme kinetics Factors affecting enzyme kinetics Effect of concentration Effect of substrate Effect of temperature Effect of pH Effect of product concentration Effect of activators Enzyme inhibition 1. Reversible inhibition. 2 . Irreversible inhibition. 3 . Allosteric inhibition Reference

3. Enzymes E nzymes are biocatalysts the catalysts of life. A catalyst is defined as a substance that increases t he velocity or rate of a chemical reaction without itself undergoing any change in the overall process. Enzymes may be defined as biocatalysts synthesized by living cells. They are protein in nature (exception - RNA acting as ribozyme ), colloidal and thermolabile in character, and specific in their action.

4. Enzyme kinetics Enzyme kinetics is the study of chemical reaction that are catalyzed by enzymes. Enzyme kinetics reveals the catalytic mechanism of that enzyme, its role in metabolism, how its activity is controlled, and how a drug or an against might inhibit the enzyme. Kinetic analysis can reveal the number and order of the individual steps by which enzymes transform substrates into products.

5. Factors affecting enzyme kinetics The contact between the enzyme and substrate is the most essential prerequisite for enzyme activity. The factors are as follows:- Concentration of enzyme Concentration of substrate Effect of temperature Effect of pH Effect of product concentration Effect of activators

6. 1. Concentration of enzyme As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases . T his property of enzyme is made use in determining the serum enzymes for the diagnosis of diseases.

7. 2. Concentration of substrate A key factor affecting the rate of a reaction catalyzed by an enzyme is the concentration of substrate, [S]. Increase in the substrate concentration gradually increases the velocity of enzyme reaction. Three distinct phases of the reaction are observed in the graph (A-linear; B-curve; C-almost unchanged).

8. Order of reaction When the velocity of the reaction is almost proportional to the substrate concentration (i.e. [S] is less than Km), the rate of the reaction is said to be first order with respect to substrate. When the [S] is much greater than Km, the rate of reaction is independent of substrate concentration, and the reaction is said to be zero order.

9. Enzyme kinetics and Km value The enzyme (E) and substrate (S) combine with each other to form an unstable enzyme-substrate complex (ES) for the formation of product (P). k1 , k2 and k3 represent the velocity constants for the respective reactions.

10. Km the Michaelis-Menten constant , is given by the formula : The following equation is obtained after suitable algebraic manipulation : where v = Measured velocity Vmax = maximum velocity , S = Substrate concentration Vm = Michaelis - Menten constant

11. Let us assume that the measured velocity (v) is equal to half of ½ Vmax . Then the equation (1) may be substituted as follows : K stands for a constant and m stands for Michaelis (in Km).

12. Km or T he Michaelis-Menten constant is defined as the substrate concentration (expressed in moles/l) to produce half-maximum velocity in an enzyme catalysed reaction. lt indicates that half of the enzyme molecules (i.e. 50%) are bound with the substrate molecules when the substrate concentration equals the Km value. Km value is a constant and a characteristic feature of a given enzyme . lt is a representative for measuring the strength of ES complex . A low Km value indicates a strong affinity between enzyme and substrate, whereas a high K. value reflects a weak affinity between them .

13. Lineweaver -Burk double reciprocal plot Lineweaver -Burk double reciprocal plot : For the determination of K, value, the substrate saturation curve V max is approached asymptotically. By taking the reciprocals of the equation (1), a straight line graphic representation is obtained . The above equation is similar to y = ax + b.

14. Therefore, a plot of the reciprocal of the velocity (1/v) vs the reciprocal of the substrate concentration (1/S) gives a straight line. Here the slope is Km /V max and whose y intercept is 1/V max . The Lineweaver -Burk plot : lt is much easier to calculate the Km from the intercept on x-axis which is -(1/Km).

15. Enzyme reactions with two or more substrates Enzyme reactions with two or more substrates :The rates of such multi substrate reactions can also be analyzed by the Michaelis-Menten approach. Enzymatic reactions with two substrates usually involve transfer of an atom or a functional group from one substrate to the other. In some cases, both substrates are bound to the enzyme concurrently at some point in the course of the reaction, forming a noncovalent ternary complex , the substrates bind in a random sequence or in a specific order. In other cases, the first substrate is converted to product and dissociates before the second substrate binds, so no ternary complex is formed.

16. Effect of temperature Velocity of an enzyme reaction increases with increase in temperature up to a maximum and then declines. A bell-shaped curve is usually observed . Temperature coefficient or Q10 is defined as increase in enzyme velocity when the temperature is increased by 10"C. For a majority of enzymes, Q10 is between c and 40c lncrease in temperature results in higher activation energy of the molecules and more molecular (enzyme and substrate) collision and interaction for the reaction to p roceed faster. The optimum temperature for most of the enzymes is between 40'C-45'C. . Majority of the enzymes become inactive at higher temperature (above 70'C).

17. Effect of pH lncrease in the hydrogen ion concentration (pH) considerably influences the enzyme activity and a bell-shaped curve is normally obtained . Each enzyme has an optimum pH at which the velocity is maximum. Below and above this pH, the enzyme activity is much lower and at extreme pH, the enzyme becomes totally inactive. Hydrogen ions influence the enzyme activity by altering the ionic charges on the amino acids substrate, ES complex etc.

18. Effect of product concentration The accumulation of reaction products generally decreases the enzyme velocity. The products combine with the active site of enzyme and form a loose complex and, thus, inhibit the enzyme activity . ln the living system, this type of inhibition is generally prevented by a quick removal of products formed .

19. Effect of activators Enzymes require certain inorganic metallic cations like Mg2+, Mn2+, zn2+, ca2+, co2*, cu2+, Na+, K+ etc" for their optimum aciivity " Rarely , anions are also needed for enzyme activity e.g. chloride ion (C11 for amylase .) Metals function as activators of enzyme velocity through various mechanisms combining with the substrate, formation of ES-metal complex, direct participation in the reaction and bringing a conformational change in the enzyme.

20. Two categories of enzymes requiring metals fbr their activity are distinguished . Metal-activated enzymes : The metal is not tightly held by the enzyme and can be exchanged easily with other ions e.g. ATPase (Mg2* and Ca2 *) Enolase (Mg2*) " Metalloenzymes : These enzymes hold the metals rather tightly which are not readily exchanged. e.g.. alcohol dehydrogenase , carbonic anhydrase , alkaline phosphatase , carboxypeptidase and aldolase contain zinc.

21. Enzyme inhibition Enzyme inhibitor is defined as a substance which binds with the enzyme and brings about a decrease in catalytic activity of that enzyme. The inhibitor may be organic or inorganic in nature. There are three broad categories of enzyme inhibition 1 . Reversible inhibition. 2. Irreversible inhibition. 3. Allosteric inhibition.

22. The inhibitor binds non-covalently with enzyme and the enzyme inhibition can be reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into two categories : l. Competitive inhibition ll. Non-competitive inhibition

23. I . Competitive inhibition : The inhibitor (l) which closely resembles the real substrate (S) is regarded as a substrate analogue. The inhibitor competes with substrate and binds at the active site of the enzyme but does not undergo any catalysis. As long as the competitive inhibitor holds the active site, the enzyme is not available for the substrate to bind. During the reaction, ES and El complexes are formed as shown:

24. competive

25. Example of competitive inhibition Antimetabolites : These are the chemical compounds that block the metabolic reactions by their inhibitory ,action 'on enzymes. Antimetabolites are usually structural analogues of substrates and thus are competitive inhibitors . They are in use for cancer therapy, Bout etc. The term antivitamins is used for the antimetabolites which block the biochemical actions of vitamins causing deficiencies, e.g. sulphonilamide , dicumarol .

26. ll. Non-competitive inhibition : The inhibitor binds at a site other than the active site on the enzyme surface. This binding impairs the enzyme function. The inhibitor has no structural resemblance with the substrate. However, there usually exists a strong affinity for the inhibitor to bind at the second site. In fact, the inhibitor does not interfere with the enzyme-substrate binding. But the catalysis is prevented, possibly due to a distortion in the enzyme conformation.

27. The inhibitor generally binds with the enzyme as well as the ES complex. The overall relation in non-competitive inhibition is represented below: For non-competitive inhibition, the Km value is unchanged while max is lowered.

28. Non compet

29. lrreversible inhibition The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. These inhibitors are usuallv toxic poisonous substances. lodoacetate is an irreversible inhibitor of the enzymes like papain and glyceraldehyde 3-phosphate dehydrogenase . lodoacetate combines with sulfhydryl (-SH) groups at the active site of these enzvmes and makes them inactive.

30. Suicide inhibition Suicide inhibition is a specialized form of irreversible inhibition. ln this case, the original inhibitor (the structural analogue/competitive inhibitor) is converted to a more potent form by the same enzyme that ought to be inhibited. The so formed inhibitor binds irreversibly with the enzyme. This is in contrast to the original inhibitor which binds reversibly.

31. Allosteric inhibition An  allosteric inhibitor   by binding to   allosteric   site alters the protein conformation in active site of enzyme which consequently changes the shape of active site. Thus enzyme no longer remains able to bind to its specific substrate.   Hence enzyme is unable to perform it's catalytic activity i.e enzyme is now inactive.

32. Reference Principles-of-Biochemistry-by- ALbert - Leningher www. Slideshare.com www. Sciencedirect.com