Module 4 - Enzymes.pptxJMLKNDWjknklcnnklMCKFIMLK

MLK x M,C KL

1. Dr. Pravin D. Patil Module 4 Enzymes

2. Enzymology Nomenclature and classification of enzymes Mechanism of action Enzyme kinetics Enzyme inhibition RNA Catalysis 2 Objectives:

3. One of the common fundamental characteristics of all living organisms is that thousands of chemical reactions occur in them at relatively low temperature . Moreover, these reactions happen at very high rates and in a highly regulated manner. Enzymes are the organic substances that act as biocatalyst and accelerate those biochemical reactions in living systems. Chemically all enzymes are proteins. Beadle (1948) defined enzymes as indispensable compounds that play a key role in metabolism by bringing direction and control to the physiological process of living cells. 3 Introduction :

4. Any change in enzyme complement of living cell is immediately reflected in the change in the physiological and biochemical process of the cell. Thus, Enzymes play a very important role in the living world. Enzymology is a branch of biochemistry which deals with the study of enzymes, their kinetics, structure, and function, as well as their relation to each other. 4 Introduction :

5. 5

6. Enzymes are generally named by adding the suffix ‘ – ase ’ to the name of the substrate on which they act. For example, the enzyme that hydrolyses starch is called amyl ase , the enzymes that hydrolyses fats or lipids are lip ase s and so on. Another way of naming the enzyme is to indicate the type of chemical reaction catalysed by that enzyme. For example, oxid ase s catalyse oxidation reaction , decarboxyl ase catalysed decarboxylation while dehydrogen ase removes hydrogen from the substrate. The International Union of Biochemistry ( IUB ) has set up an enzyme Commission for working out a systematic classification and nomenclature of all enzymes . 6 Nomenclature and classification of enzymes :

7. 7 Nomenclature and classification of enzymes :

8. 8 Nomenclature and classification of enzymes :

9. Oxidoreductase: These enzymes catalyse oxidation-reduction of two substrates. In this reaction, one substrate get oxidized while another gets reduced. e.g. Dehydrogenase enzyme Transferase: These enzymes catalyse the transfer of chemical group (such as aldehydic, ketonic, alkyl, methyl etc.) from one molecule to the other. e.g., Transaminase enzyme transfers amino group, transmethylase transfers methyl group, transaldolase transfers aldehydic group, etc. 9 Nomenclature and classification of enzymes :

10. Hydrolase: These enzymes catalyse hydrolysis of substances which result in cleavage of that substrate. e.g. Esterase catalyses hydrolysis of esters, glycosidase break glycosidic linkage. Lyases: Enzymes of this group involved in the removal of groups from substrates by mechanisms other than hydrolysis and double bond is introduced at the place of removal of that group. e.g. Lyases act on C-C, C-O, C-N, C-S bonds. In most of the cases, lyases enzyme requires coenzymes. e.g., aldolase, decarboxylase etc. 10 Nomenclature and classification of enzymes :

11. Isomerase : These enzymes catalyse the reactions where intramolecular rearrangement take place in the substrate and isomers are formed. Many enzymes involved in carbohydrate metabolism are isomerase enzymes. e.g. triose phosphate isomerase, bisphosphoglycerate mutase, and photoisomerase . Ligases : Ligases catalyse the linkage of two molecules often coupled with the cleavage of ATP or similar compounds. They are also called synthetase. e.g. DNA lygase 11 Nomenclature and classification of enzymes :

12. The enzymes act on a substrate by combining with the substrate molecules to form an enzyme-substrate complex. Enzymes ( E ) have specific active sites for the attachments of substrate ( S ) molecule where an enzyme can form intimate relationship with the substrate. It is presumed that the enzyme-substrate combination ( ES ) brings about a deformation in some of the bonds in substrate molecule which favours the reaction to produce a product. 12 Mechanism of action:

13. The binding of enzyme and substrate is highly specific. A given enzyme usually binds to only one kind of substrate. The specificity of enzyme-substrate interaction arises mainly from hydrogen bonding and shape of the active site (which rejects molecules that do not have sufficiently complementary shape). Two important models (theories) have been proposed to describe the mode of action of enzymes viz., lock and key mechanism and induced fit mechanism . 13 Mechanism of action:

14. This theory was proposed by Fisher (1894). According to this theory, the formation of enzymes-complex during enzyme action is analogous to the fitting of lock and key. It is believed that the enzyme and the substrate both have strictly complementary structures which during complex formation fit to each other like a specific key in a particular lock. Here the enzyme is like a lock and substrate is like a key. Only a specific key (substrate) with correctly positioned teeth fits into the keyhole (active site) of the lock (enzyme). 14 Mechanism of action: lock and key mechanism

15. Thus, the active site of the enzyme is a rigid and pre-shaped template where only a specific substrate can bind. In simple words as a particular lock can be opened by a particular key, in a same way particular enzyme acts on a particular substrate. This theory gave a satisfactory explanation for enzyme action, however, this theory fails to explain the flexible nature of an enzyme 15 Mechanism of action: lock and key mechanism

16. This model was proposed by D. Koshland in 1966. According to this model, the enzyme and substrate do not have strictly complementary structures but the enzyme has flexible active site with changes according to substrate configuration. 16 Mechanism of action: Induced fit model

17. Thus, the enzyme show induced fit mechanism during enzyme-substrate complex formation. This model can be compared with hand and glove. The same glove can be fit in the hands of many persons. 17 Mechanism of action: Induced fit model

18. In other words, the glove adjusts itself in the hands of those persons who wear it. Similarly conformational changes in the enzyme are induced by the substrate during enzyme action 18 Mechanism of action: Induced fit model

19. 19 Mechanism of action: Induced fit model

20. 20 Mechanism of action:

21. 21 Module 5: Enzymes (Introduction)

22. All chemical reactions require some energy input to begin. The amount of energy needed for a reactant to undergo a chemical reaction is called activation energy. Energy is needed to break existing bonds before new bonds can be formed. The formation of new bonds may release more energy than was needed to break the original bonds. Even though there may be a net release of energy, the need for activation energy can act as a barrier to the chemical reaction occurring. 22 Energy of activation in the catalytic action of an Enzyme:

23. 23 Energy of activation in the catalytic action of an Enzyme:

24. 24 Energy of activation in the catalytic action of an Enzyme:

25. Enzymes lower the barriers that normally prevent chemical reactions from occurring by decreasing the required activation energy. The initial rise in energy is the energy input needed before the reaction will occur (activation energy). The subsequent drop in energy is the energy released by the reaction. From the graph given below, it is clear that the reaction requires less activation energy when an enzyme is present. This is why the addition of an enzyme allows a reaction to proceed at a much faster rate. 25 Energy of activation in the catalytic action of an Enzyme:

26. Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a substance might inhibit the enzyme. 26 Enzyme kinetics and kinetic parameters:

27. Michaelis and Menten (1913) while working on hydrolysis of sugar by the enzyme invertase, first who proposed the equation for enzyme kinetics to explain the mode of action of enzyme. They assumed that the enzyme has single substrate and a single product. However, the equation is applicable to all the enzyme reactions. 27 Enzyme kinetics and kinetic parameters:

28. As enzyme- catalysed reactions are saturable, their rate of catalysis does not show a linear response to increasing substrate. If the initial rate of the reaction is measured over a range of substrate concentrations (denoted as [S]), the initial reaction rate (V ) increases as [S] increases. However, as [S] gets higher, the enzyme becomes saturated with substrate and the initial rate reaches V max , the enzyme's maximum rate. 28 Enzyme kinetics and kinetic parameters:

29. Michaelis and Menten’s equation describes how the (initial) reaction rate V depends on the position of the substrate-binding equilibrium and the rate constant. 29 Enzyme kinetics and kinetic parameters:

30. In the above equation, V is the velocity of the reaction, and K m is known as Michaelis’ constant. Michelle’s constant ( K m ) is numerically equal to the substrate concentration when the reaction proceeds at half its maximum rate and is expressed in moles/ liter. It shows that a high substrate concentration is necessary to attain half saturation or V max , and the enzyme has low affinity for the substrate. 30 Enzyme kinetics and kinetic parameters:

31. Practical significance of kinetic parameters: The study of enzyme kinetics is important for two basic reasons. Firstly, it helps explain how enzymes work , and secondly, it helps predict how enzymes behave in living organisms . The kinetic constants defined above, K M and V max , are critical to attempts to understand how enzymes work together to control metabolism. 31 Enzyme kinetics and kinetic parameters:

32. A number of compounds have ability to combine with certain enzymes and thereby block the reactions. Such compounds are termed as inhibitors and the process is termed as inhibition. The enzyme inhibition can be irreversible or reversible. 32 Enzyme inhibition:

33. 33 Enzyme inhibition: Reversible and Irreversible Inhibition

34. These inhibitors usually bound covalently to the enzyme and destroy the functional group in active site. Most of the irreversible inhibitors are toxic substances. For example, penicillin acts as an irreversible inhibitor for the enzyme glycoprotein peptidase. This enzyme plays an important role in the bacterial wall synthesis. Once the cell wall synthesis is blocked, the bacteria cells undergo lysis and the growth is stopped. 34 Irreversible inhibition:

35. In this case, the inhibitor can dissociate from the enzyme as the former shows non-covalent bonding with enzymes. There are three types of reversible inhibitors as follows: 35 Reversible inhibition:

36. Competitive inhibitors: The structure of these inhibitors closely resembles that of the normal substrate. Because of similar structure, competitive inhibitor binds to enzymes active site. For example succinic acid dehydrogenase which is an enzyme of for succinic acid inhibited by malonic acid having similar structure. 36 Reversible inhibition:

37. Non-competitive inhibitors: These inhibitors bind to the enzyme at the site other than its active site. This binding alters the enzyme’s conformation and thereby blocks the reaction. Non-competitive inhibitors can be attached to free enzyme or enzyme- substrate complex. It prevents the breakdown of ES Complex completely or may reduce the rate of breakdown. A variety of metal ions such as Ag+2, Hg+2 Pb+2 act as non-competitive inhibitors. 37 Reversible inhibition:

38. Uncompetitive inhibitors: In this type the innovative combines with only ES Complex but not with free enzyme. Similar to non-competitive inhibition, uncompetitive inhibition is not reversed by increasing the substrate concentration. This type of inhibition is rare with single substrate and often found in enzymatic reactions with two or more substrates. 38 Reversible inhibition:

39. Enzymes, nature's biocatalysts, have found diverse and invaluable applications in various industries. Their ability to accelerate chemical reactions under mild conditions has revolutionized industrial processes, offering sustainability, efficiency, and cost-effectiveness. Let's explore some industrial applications of enzymes with a focus on a detailed example. 39 Industrial Applications of Enzymes:

40. 40 Industrial Applications of Enzymes:

41. 41 Industrial Applications of Enzymes:

42. 42 Thank you…