Enzymes

Introduction, Nomenclature of enzymes, Classification of enzymes on the basis of site of action, on the reaction of catalysis and Classification depends upon substrates on they which act, Specificity of Enzymes, Active Site of An Enzyme: 1. Lock-key model 2. Induce fit model, Factors Affecting Enzymes Reaction, Enzyme 1.Inhibition Competitive inhibition, 2. Non-Competitive inhibition, Isoenzymes, Allosteric Enzymes, Co-Factors, Turnover Number of An Enzyme, Pharmaceutical Importance Of Enzymes,

1. Enzymes Presented By Ms. Monika Maske Assistant Professor M. Pharm ( Pharmaceutical Chemistry) 1

2. Introduction Enzymes increases the rate of reaction, without altering its equilibrium. They are initially produced in cell an some are excreted through cell wall and function in the surrounding environment. Enzymes are proteins that act as catalysts for biochemical reactions. 2

3. Almost all biochemical reactions require catalysts . Recently, observed that some RNA molecules have catalytic activity. The term first introduced by KUHNE in 1878. 3

4. Nomenclature of Enzymes The word enzyme is formed from two Greek words: E n means inside and zyme , which means yeast i.e., the word enzyme means inside yeast . There are many methods for naming enzymes: 1- The old trivial name as pepsin and trypsin. 2- The name of substrate and the suffix – ase added to it as lactase acting on lactose . 4

5. 3- Two words , one for the substrate and the other for the type of reaction e.g. Succinate dehydrogenase, pyruvate decarboxylase and glutamine synthetase . Enzymes are generally named according to the reaction they catalysed or by suffixing “ ase ” after the name of substrate. The International Union of Biochemistry and Molecular Biology developed a nomenclature for enzymes. 5

6. Each enzyme described by a sequence of four numbers preceded by “EC”. EC denotes Enzyme Commission and the no. of enzyme is called EC number. When classified, each enzyme is assigned the Ec number, in the form of digits separated by dots. 6

7. The first digit denotes the class (reaction type) of the enzyme. second digit denotes the functional group (subclass) upon which the enzyme acts. The third digit denotes the coenzyme(sub-subclasses). The fourth digit denotes the substrate (serial no. identify enzyme within sub-subclass). 7

8. The class, subclass and sub-subclass provide additional information about the reaction classified. The last printed list of enzymes appeared in year 1992. since then it has been updated and maintained online. 8

9. 9 Sr. No Name of substrate or Reaction Enzyme 1 Urea Urease 2 Maltose Maltase 3 Protein Protease 4 Carbohydrate Carbohydrase 5 Reduction Reductase 6 Group transfer Transferase 7 Isomerisation Isomerase

10. Classification of Enzymes The classification is based on the reaction of catalysis. Oxido -reductases: Enzymes catalyse redox reactions, i.e. the removal of hydrogen or addition of oxygen. - These reaction catalysed by enzyme succinate dehydrogenase. 10

11. 2. Transferases: It transfer groups like –NH3, -CH3, -CH2OH. These carried out by transferase enzymes. It transfers the amino group from an amino acid to alpha-keto acid. 3. Hydrolases: These enzymes catalyze the hydrolytic cleavage reaction. 11

12. 4. Lyases: These enzymes catalyse the cleavage reactions which are non-hydrolytic. Ex. Aldolase, histidase etc. 5. Isomerases: These enzyme responsible for the isomerization reaction. These reaction requires dextrose isomerase enzyme. Dextrose Fructose 12

13. 6. Ligases: These enzymes catalyse the formation of bonds. ex. DNA ligase 13

14. Classification On The Basis of Site of Action Endoenzymes- Which act only inside the cell are known as endoenzymes or intracellular enzymes. It involves the synthesis of cell components, food reserves & bioenergetic i.e liberation of energy from food. Ex. Isomerases, Phosphorylases. 14

15. b) Exoenzymes- The enzymes which are secreted outside the cell are known as exoenzymes or extracellular enzymes. These are normally digestive in their function. They hydrolyse very complex molecules into simple compounds i.e. proteoses, lypases acting on proteins, lipids respectively. 15

16. c) Constitutive Enzymes: These enzymes are produced in absence of substrate are known as constitutive enzymes. Which produced in constant rate, in constant amounts of metabolic state of organism. These are part of basic & permanent enzymic action of cell. Ex. Enzymes of glycolytic series. 16

17. d) Induced Enzymes: These are present in trace amounts but their conc. Gets immediately increased in presence of substrate on which they act. Microorganisms produce them in response to the presence of substrate in the environment only known as induced enzymes. Ethanol, barbiturates are powerful in inducing hepatic microsomal enzymes. 17

18. Classification of Enzymes Classification depends upon substrates on they which act. Amylolytic enzymes or Carbohydrases : Enzymes which act on only on carbohydrates known as amylolytic or carbohydrases . 1. Amylopsin or Diastase 2. Ptyalin 3. Malt-diastase 4. Invertase 18

19. 5. Maltase 6. Zymase 7. Emulsin 8. Myrosin 9. Hemicellulose 10. Hyaluronidase 19

20. B . Proteolytic Enzymes or Proteases: This enzymes act on proteins & hydrolos e them. 1. Trypsin 6. Fibrinolysin 2. Chymotrypsin 7. Eurokinase 3. Pepsin 8. Streptokinase 4. Renin 9. Papain 5. Muramidase 10. Bromelain 20

21. 3. Lypolytic Enzyme: Enzymes which act on fats or lipids. 1. Lypase 2. Steapsin 3. Pancreatin 4. Fungal lipase 21

22. Enzyme Catalysed reactions The reactants, which are transformed in an enzymic reaction are called the substrates. During enzymic reaction the substrate is converted into a product. Let us consider a general reaction; 22

23. E + S K1, K2 ES K3 E + P Where, [E]- Enzyme Conc. [ES]- Enzyme substrate complex conc. [S]- Substrate conc. [P]- Product conc. While, K1, K2 and K3 are equilibrium constants. 23

24. Enzymes increases the rate of biochemical reaction by reducing the energy of activation , without shifting the chemical equilibrium The substrate is converted to product. The reaction is carried out in absence of enzyme, the energy of activation is high. In presence of enzyme, the energy of activation is reduced. 24

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26. Specificity Of Enzymes Enzymes are highly specific in their action. Inorganic catalysts are not specific in their action. It is important feature of enzymes. Man-made catalysts, they vary in their degree of specificity. Some have absolute specificity. 26

27. Ex. Aspartate is an example of absolute specification. Aspartate acts only on fumarate to produce L-aspartate. Some enzymes can act on a wide variety of substrates, they are structurally related. These types of enzyme have broad specificity. Aspartates converts fumarate & not to D-aspartates. 27

28. Active Site of An Enzyme It is a portion of an enzyme, to which the substrate binds & gets converted into the product. The substrate binds with enzyme with the help of weak ionic interactions like H-bonds. Conformation of active site:- The binding of a substrate with an enzyme at the active site, it explain by 2 models. 1. Lock-key model 2. Induce fit model 28

29. Lock-key model- The shape of active site and substrate is complementary to each other. The substrate molecule fits into the site of an enzyme just as key fits into a lock. This model shape of active site is rigid & complementary to the shape of substrate molecules. These model cannot explain the broad specificity of some enzyme. 29

30. B) Induce Fit Model- In this modal the active site of an enzyme is considerably flexible. So wide variety of substrate molecules. The shape of active site is ,made complementary to the substrate to certain extent thus, the substrate binds with the enzyme hence, called as induced fit model. 30

31. Factors Affecting Enzymes Reaction Effect of substrate concentration (Michaelis-Menten Equation)- Keep enzyme conc . is constant, increase in the conc. of substrate , results the rate of reaction increases. The initial stage, rate is directly proportional to the substrate conc. If substrate conc. further increased, velocity of reaction remains unchanged. 31

32. Enzymes are completely saturated with the substrate, the velocity at this stage is called the maximum velocity denoted by V or V max. Rate equation of enzyme catalysed reaction K m called the Michaelis Menten constant. 32

33. It define as substrate conc. At which the velocity of an enzyme catalysed reaction is exactly half of V or V max. 33

34. The rate of equation of an enzyme catalysed reaction can be transformed to its inverse form & is called the Linweaver Burk equation. 34

35. 2. Enzyme Concentration – Enzymic activity is directly proportional to the Conc. Of enzyme. Enzymes are usually present in very low molar conc. 35

36. 3. Effect of pH- Most enzymes have a characteristics pH at which their activity is maximum. Going below & above this pH, the activity declines. This characteristic pH is called the optimum pH of an enzyme. pH activity relationship of any enzyme depends on the acid-base properties of enzyme & substrate, and on other factors. 36

37. It is not necessary that optimum pH of enzyme identical to normal intercellular pH. Trypsin has optimum pH about 8 hence, buffer is needed for action of enzymes to maintain the pH. 37

38. 4. Effect of Temperature- The rate of enzyme catalysed reactions increases as the temperature increases upto the optimum temp. Further rise in temp. decreases the velocity of reaction. The temp. at which enzyme shows maximum active. Further rise in temp. results in inactivation of enzyme called as thermal denaturation. 38

39. 5. Effect of Inhibitors – Presence of enzyme inhibitors reduces the enzyme action. Heavy metals are the inhibitors for enzyme activity. 6. Effect of Activators – Presence of activators in certain conc. Increases the enzyme activity. Cysteine HCl increases the proteolytic activity of papain. 39

40. 7. Effect of Radiation & Light – Some enzymes may be deactivated by exposure to UV, beta, gamma and X-rays. Ex. Salivary amylase is deactivated by exposure to UV rays. 40

41. Enzyme Inhibition The functional grp. Of enzymes, from their active site & surfaces, react with a varity of chemical reagents & their catalytic activity is reduced. These reagents are called inhibitors and process is called enzyme inhibition. Some are non-physiological in origin. These inhibitors regulate the activity of enzymes. According to mode of action , it classified as 1. Competitive inhibition 2. Non-Competitive inhibition 41

42. 1. Competitive inhibition A competitive inhibitor often has structural features similar to those of the substrate whose reactions they inhibit. The inhibitor and the substrate both are competing for the active site of an enzyme it is called competitive inhibition. 42

43. 2. Non-Competitive inhibition These are not influenced by the conc. of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. They also bind with the same affinity to the free enzyme & form the Enzyme-Substrate complex . It change the shape of enzyme & active site. 43

44. Isoenzymes The enzymes which have multiple molecular forms in the same organism, catalysing the same biochemical reaction are called isoenzymes. Lactate + NAD + Pyruvate + NADH+ H + These reaction is catalysed by enzyme lactate dehydrogenase which is present in five different molecular forms in the tissues. All have different amino acid composition & sequence. 44

45. Allosteric Enzymes These are regulatory enzyme. The catalytic activity is regulated by itself. The regulation is mediated via specific metabolites. These metabolites are called allosteric modulators. Allosteric enzymes have allosteric site, in addition to the active site to which allosteric modulator binds. 45

46. 2 types of allosteric modulators, Positive allosteric modulators if they increase the activity of an enzyme. Negative allosteric modulators which reduces the activity of an enzyme. 46

47. Co-Factors Some enzymes requires a non-protein moiety for catalytic activity which is called as a co-factor. Some co-factors derived from vitamins they called as co-enzymes. Co-enzymes are low mol. wt. organic substances, derived from vitamins. Ex. Pyridoxal phosphate is derived from vit. B6 which is a co-enzyme of transaminase. 47

48. There are some enzymes which require metal ions for their catalytic activity called as metal co-factor. Ex. Zinc is required for carboxypeptidase. Enzyme activity units Amounts which causes transformation of 1.0 μ mole (10 6 ) of substrate per minute at 25 C, under optimal condition of measurement. Specific activity defined as the no. of enzymes unit per milligram of protein. 48

49. Turnover Number of An Enzyme It is the number of substrate molecules transformed per minute by a single enzyme molecule when the enzyme conc. is the rate limiting factor. Feed back inhibition:- In some enzymes, their activity is inhibited by their product or the successive metabolites of the metabolic pathway called as feed back inhibition. The metabolite D inhibits enzyme E1 this called feed back inhibition. 49

50. Pharmaceutical Importance Of Enzymes Medicinal significance of enzymes:- The mode of action of enzymes explains the action of certain drugs which are enzyme inhibitors, these types of drugs belongs to class antimetabolite Allopurinal is used in treatment of gout, it prevents the formation of uric acid. It is competitive inhibitors of xanthin oxidase. 50

51. 2. Enzyme Therapy:- Enzymes can used for treatments like tumours, lactose in tolerance in children. 3. Manufacturing of Bulk Drugs:- Some enzymes used for mfg. of bulk drug like penicillin acylase for production of 6-amino-penicilanic acid (6 AP). Urokinase in cardiac diseases, streptokinase in treatment of thrombosis. 51

52. 4. Diagnostic Uses of Enzymes:- Enzymes are known as marker of cellular damage. The amount of enzymes present in the plasma is used for investigation of diseases of liver, heart, skeletal muscle, etc. Aspartate amino transfererase and lactate dehydrogenase are seen in cardiac and hepatic tissues. Trypsin enzyme seen in stomach causes liver diseases and malnutrition. 52

53. 5. Medicinal and Clinical uses:- Enzymes can be used for Aiding Digestion. Ex. Amylases, Lipase and proteases. Used as Deworming agents. Ex. Papain. Enzymes act as anti-clotting agents like fibrinolytic and thrombolytic. Ex. Urokinase, streptokinase. Can be used as surface disinfectants. Ex. Trypsin. 53

54. Used in the diagnosis purpose. Ex. Glucose oxidase along with peroxidase to detect the level of glucose. Liver disease: SGPT, SGOT Heart attacks (MI): SGOT 6. Industrial Purpose:- Enzyme can be used in textile industry. Used for leather purpose. 54

55. Used in paper manufacturing, organic compounds manufacturing like lactic acid. 7. Enzymes as food and in Food Industry:- Enzymes can be used in the meat packing industry. 8. Research:- Several enzymes are used for detection of biochemical reactions. 9. Beverage industry:- Papain is used to stabilize chill proof bear. Yeast enzymes are used in beverage industry. 55

56. Thank You 56