Enzymes

1. Enzymes

2. Definition of enzyme Enzymes are the biocatalysts synthesized by living cells which have specificity in their action. They are protein in nature and are colloidal and thermolabile.

3. Enzymes may be • Intracellular: they are functional inside the cells. e.g. hexokinase, pyr vate dehydrogenase etc. • Extracellular: they are functional outside the cells. e.g. digestive enzymes

5. Classification of enzymes

6. Oxidoreductases:oxidation reduction • AH2 + B ------- A + BH2 • Transferases:group transfer • A-X + B -------- A + B-X • Hydrolases:hydrolysis • A-B + H2O -------- AH + BOH • Lyases:addition-elimination • A-B + X-Y -------- AX-BY • Isomerases:interconversion of isomers • A -------- A’ • Ligases:condensation (ATP dependent) • A + B------------ A-B

7. Chemical nature • The functional unit of enzyme is called holoenzyme which is made up of apoenzyme (protein part) and coenzyme (non-protein part). Holoenzyme ---------- apoenzyme + coenzyme (active enzyme) (protein part) (non-protein part) • If the non protein moiety is tightly bound to apoenzyme it is known as prosthetic group

8. Type of enzymes • Monomeric enzymes: made up of single polypeptide e.g. ribonuclease, trypsin etc. • Oligomeric enzymes: enzymes that possess more than one polypeptide chain e.g lactate dehydrogenase, aspartate transcarbamoylase etc. • Multienzyme complex: group of enzymes or sites that carries different reactions in a sequence. It is active only in intact complex not individually e.g. pyruvate dehydrogenase, fatty acid synthase etc.

9. Mechanism of enzyme action

11. How enzyme works?

12. Effect of enzyme

13. Factors affecting enzyme activity • Effect of enzyme concentration : • Reaction velocity ↑ as enzyme concentration ↑ • Effect of product concentration: • Reaction velocity ↓ as product concentration ↑

14. Effect of pH: bell shaped curve

15. Effect of temperature: • bell shaped curve, maximum at 37ºC • Effect of activators: • Metallic cations may present in some enzymes so enzymes may be either metal activated enzymes & metalloenzymes • Effect of time: • Variation in time due to change in pH & temperature

17. Effect of substrate concentration: • Increase in substrate concentration gradually increases the reaction velocity of the enzyme. • At low substrate concentration • velocity α substrate level • At medium substrate concentration • velocity is ≈ α substrate level • At high substrate concentration • velocity is independent to the substrate level

21. Michaelis-Menten equation graph

23. Lineweaver-Burk double reciprocal plot

24. Inhibition of enzymes • Reversible inhibition • Competitive inhibition • Noncompetitive inhibition • Uncompetitive inhibition • Irreversible inhibition • Allosteric inhibition

25. Competitive inhibition • Inhibitor closely resembles the real substrate i.e. substrate analogue, competes with the substrate and binds in the active site of the enzyme • In competitive inhibition Km value increases whereas Vmax remains unchanged. • Example: succinate dehydrogenase with malonic acid inhibitor, xanthine oxidase with allopurinol inhibitor

26. Competitive inhibition

27. Noncompetitive inhibition • Inhibitor binds at a site other than active site on the enzyme surface • In noncompetitive inhibition Km remains unchanged whereas Vmax decreases. • Example: heavy metal ions (Ag+, Pb++, Hg++ etc.) noncompetitively binds with enzyme

28. Noncompetitive inhibition

29. Uncompetitive inhibition • Inhibitor doesn't bind with enzyme but only binds with enzyme substrate complex • Uncompetitive inhibition decreases both Km and Vmax values of the enzyme.

30. Uncompetitive inhibition

31. Irreversible inhibition • Inhibitors bind covalently with the enzymes and inactivate them which is irreversible. • Iodoacetate is an irreversible inhibitor of the enzymes like papain & glyceraldehyde-3-phosphate dehydrogenase • Many organophosphorus insecticides like melathion are toxic as they block the activity of acetylcholine esterase.

32. Allosteric inhibition • Some of the enzymes possess additional sites known as allosteric sites besides the active site. These enzymes may be activated or inhibited according to the allosteric effector.

33. Regulation of enzyme activity • Regulation of enzyme activity is acquired by one or more of the following ways. • Allosteric regulation: • Activation of latent enzymes: • Compartmentation of metabolic pathways: • Control of enzyme synthesis: • Enzyme degradation: • Isoenzymes:

34. Allosteric regulation • Some of the enzymes possess additional sites known as allosteric sites besides the active site. These enzymes are regulated according to the allosteric effectors • (+)ve & (-)ve allosteric effecters may bind to the allosteric site of enzyme to increase or decrease the effect of enzyme. • K- class of allosteric enzymes: effector changes the Km but not Vmax e.g. phosphofuctokinase • V-class of allosteric enzymes: effector alters the Vmax, and not the Km e.g. acetyl CoA carboxylase • Feed back regulation: process of inhibiting the first step by the final product in a series of reactions.

35. Activation of latent enzymes • Latent or inactivated enzymes become active by its modification. • Proenzymes become active by the breakdown of one or more peptide bonds e.g. pepsinogen, trypsinogen, plasminogen etc • Certain enzymes exist in the active and inactive state and they are interconvertible e.g. covalenbt modification, phosphorylation & dephosphorylation and oxidation and reduction. • Certain enzymes are activated by the help of hormones through the mediation of cAMP.

36. Compartmentation of metabolic pathways • Some enzymes occur only in the specific organelle of cell and function there only regulating their function. • Synthetic and breakdown pathways are operative in different cellular organelles. Enzymes for fatty acid synthesis are found in cytosol but enzymes of fatty acid degradation are found only in mitochondria

37. Control of enzyme synthesis • Synthesis of enzymes is regulated by genes by the mechanism of induction and repression through the mediation of hormones. • Insulin and glucagon regulate the enzyme function by induction and repression • There are two types of enzymes: constitutive & adaptive

38. Enzyme degradation • Enzymes are also regulated by degrading after their function. Different enzymes have different half-lives ranging from minutes, hours to days • In general key and regulatory enzymes are degraded most rapidly and when required synthesized quickly.

39. Isoenzymes • The multiple forms of the same enzyme catalyzing the same reaction but having different physical and chemical properties are known as isoenzymes. • Many of them are tissue specific but differ in Km, Vmax or both • LDH has 5 isoenzymes • LDH1, LDH2, LDH3, LDH4 & LDH5 • CK has 3 isoenzymes • CK1, CK2 & CK3

40. Units of enzyme activity • Never expressed in terms of their concentration (mg or µg), but are expressed only as activities. • Katal: one katal denotes the conversion of one mole substrate per second • IU: one IU denotes the conversion of one micromol of substrate per minute. • IU = 60 µkatal

41. Applications of enzymes • DIAGNOSTIC USE • THERAPEUTIC USE • ANALYTICAL USE • GENETIC ENGINEERING • INDUSTRIAL USE

42. DIAGNOSTIC: • CPK, AST, LDH in myocardial infraction • ALT, AST, ALP in liver disease • CPK, Aldolase, AST in muscular dystrophy • Acid phosphatase, in cancers β-glucuronidase, transaminase LDH etc. • THERAPEUTIC: • Streptokinase to remove blood clots • α-Antitrypsin to treat emphysema • ANALYICAL:for estimation of • Glucose peroxidase Glucose • Urease Urea • Uricase Uric acid • Lipase Triglyceride • ALP/horse radish proxidase ELISA • GENETIC ENGINEERING: • Restriction endonuclease gene transfer, DNA fingerprinting • Taq DNA polymerase PCR • INDUSTRIAL: • Rennin Cheese preparation • α-Amylase Food industry

43. Lactate dehydrogenase (LDH) • Tetrameric enzyme with 4 polypeptide subunits (M and H) • 5 ISOENZYMES • LDH-1 (4H) - Heart • LDH-2 (3H 1M) - Reticuloendothelial system, RBC • LDH-3 (2H 2M) - Brain, Kidney, lungs • LDH-4 (1H 3M) - Liver • LDH-5 (4M) - liver and striated muscle • Normal in serum: LDH-2 (H3M) > LDH-1 (H4). • In MI: Reversed= LDH-1 (H4) > LDH-2 (H3M) called flipped pattern

44. Creatine kinase (CK) • Creatinine phosphokinase [CK or CPK] is a Dimeric enzyme composed of two subunits M (Muscle) & B (Brain) • 4 ISOENZYMES: • 3 cytosolic isoenzymes - CK-3, CK-2, CK-1 • 1 mitochondrial isoenzymes – CK-Mt • CK ISOFORMS- CK-MB1, CK-MB2 and CK-MM1, CK-MM2, CK-MM3 • Normally: CK-2 MB < 6% of total CK • In MI: CK-2 MB ≥ 6% of total CK: most specific indicator of myocardial damage • CK is elevated in • Myocardial infarction [MI] – CK total and CK-MB ↑ • Muscular Dystrophy, Muscular Injury, Fracture

45. ENZYMES IN LIVER DISEASE

46. Enzymes in myocardial infraction • LDH: Rises (LDH1) within 12-24 h, peak at 48-72 h, and remain elevated for >7 days. • CK: Rises (CK2) within 4 to 8 h, peak at 24 hr, and return to normal by 36 to 72 hrs. • AST: Rises within 6-8 h, peak at 24-48 h, and return to normal by 4-6th day.