ENZYMES

1. ENZYMES Dr Nithin Kumar U Assistant professor Biochemistry Yenepoya medical college

2. Enzymes: Basics • Rate of a Reaction • Reversible & Irreversible Reactions • Reaction Equilibrium • Catalysis

3. Background: A reaction r1 A+B C+D • Reaction rate: r1α[A] [B] • Therefore, r1 = k1 [A] [B]

4. Reversible reaction r1 (k1) A+B C+D r2(k2) • forward reaction (left to right) • backward reaction (right to left) • state of equilibrium – chemical equilibrium. • At equilibrium, r1 = r2

5. Background • r1= k1 [A] [B] & r2= k2 [C] [D] • At equilibrium, k1[A] [B] = k2 [C] [D] [A] [B] k2 [C] [D] k1 • Law of mass action - for reversible reactions (Effects of [S]and [P] on the direction in net reaction proceeds) = Keq(equilibrium constant) =

6. Background [A] [B] [C] [D] • Freely reversible reaction, K eq value is 1 There is no energy change (ΔG = 0); • Irreversible reaction K eq value very high (endergonic reaction; ΔG = +ve ) or Negligible(exergonic reaction; ΔG =–ve). Keq=

7. Catalysis • Catalyst increases the rate of a chemical reaction , remains unchanged chemically at the end of the reaction • Phenomenon is CATALYSIS • neither cause chemical reactions to take place nor change the equilibrium constant of chemical reactions

8. Catalysis • Catalyze forward & backward reactions equally • Only catalyze reaction in thermodynamically allowed direction • Catalysts accelerate chemical reactions by lowering the activation energy or energy barrier

9. Effect of catalyst on activation energy of a reaction

10. Enzymes: Definition • Biocatalysts synthesized by living tissues which increase the rate of reaction without getting consumed in the process

11. Enzymes: Introduction • Biocatalysts • Neither consumed / permanently altered • Colloidalorganic compounds – proteins • Formed by living organisms • High specificity for their substrates and reaction types

12. Enzymes: Introduction • No formation of unnecessary by-products • Function in dilute aqueous solutions under mild conditions of temperature and pH • Physiological regulation • Several enzymes can work together in a specific order creating - metabolic pathways

13. Enzymes: Medical importance • Accelerate 106 to 1012 times • Regulatory enzymes sense metabolic signals • Inherited genetic disorders(Phenylketonuria) • Inhibitors of enzymes can be used as drug Ex: Lovastatin for HMG CoA reductase • Clinical enzymology

14. Enzymes: History • En-zyme = in yeast • In 1850s Louis Pasteur – “ferments” – fermentation of sugar into alcohol by yeast • Urease – first enzyme to be isolated in crystalline form in 1926 • Ribozymes ( made up of RNA)

15. Active site • Size of enzymes >substrates • “small region at which the substrate binds and catalysis take place” • Imparts efficiency: -Local concentration -shields substrate from solvent • Situated in a pocket or cleft of the enzyme • The active site contains - -substrate binding site (substrate) -catalytic site (reaction)

16. Active site • Catalytic sites of enzymes contain sites for binding cofactors or coenzymes • exists d/t tertiary structure of protein loss of native enzyme structure derangement of active site loss of function • For catalysis to take place substrate/s should bind to the substrate binding site reversiblyby weak non-covalent bonds (Hydrogen bond, Van der walls force, hydrophobic interactions)

17. Active site • Not rigid in structure and shape • Flexible to promote the effective binding of substrate to the enzyme • Responsible for substrate specificity • If an enzyme is denatured or dissociated into subunits catalytic activity is lost • Enzymes acts within the moderate pH and temperature

18. Active site • In active site 3–4 amino acids directly involved in catalysis- catalytic residues • Amino acids far away in the primary structure contribute to the formation of active site • amino acids at the active sites – *serine *aspartate *histidine *cysteine *Lysine *arginine *glutamate *tyrosine

19. Active site

20. Enzymes andEnzyme Catalyzed Reactions • Substrate: Reactant/s on which the enzymes act to catalyze the reaction • Enzymes are much larger than the substrates they act on

21. Holoenzyme, Apoenzyme • Some enzymes contain a non protein prosthetic group other than protein component • Holoenzyme=Apoenzyme+cofactor (protein) (non protein)

22. Cofactors • Non-protein factors required for catalysis • Bind to the catalytic site • organic or inorganic • Organic cofactors -prosthetic groups -- tightly bound -coenzymes – released from active site during reaction • Inorganic cofactors – activators • Exceptions -- FMN ,FAD, and biotin are tightly bound to enzymes. but called as coenzymes.

23. Cofactors • Exceptions -- FMN ,FAD, and biotin are tightly bound to enzymes, but called as coenzymes

24. Prosthetic group • tightly bound to enzyme by covalent bond • cannot be separated from enzyme by Dialysis • Ex -Biotin in carboxylases • Apoenzyme + Prosthetic group = Holoenzyme (active enzyme)

25. Coenzymes • derivatives of vitamins • Boundreversibly by weak non-covalent bonds to active site and released during the reaction • separated easily from enzyme by dialysis • Affinity for the enzyme is similar to substrate • chemically changedby catalysis • considered as co-substrate • Function: carriers of various groups

26. Coenzymes • Hydrolases (class 3) - not require coenzymes • IUB classes: I,II,V and VI need coenzymes

27. Inorganic Cofactor/Activators • Metals • 2 types - Metal activated enzyme - Metallo enzymes metal activated enzymes • metal is not tightly bound by the enzyme • Ex - *ATPase (Mg2+) *Enolase(Mg2+) *Chloride (Cl-) -salivary amylase *Ca2+ and Pancreatic lipase

28. Inorganic Cofactor/Activators Metallo enzymes • Metals are tightly bound with enzymes • Ex - *alcohol dehydrogenase (zinc) *carbonic anhydrase (zinc) *DNA Polymerase (zinc) *Xanthine oxidase(molybdenum) *Catalase, peroxidase(Iron) *Cytochrome oxidase(iron)/(copper) *Hexokinase, pyruvate kinase(Mg2+) *Glutathione peroxidase( selenium)

29. Mechanism of enzyme action • Enzymes act by binding substrates & loweringactivation energy (energy needed for reactants to undergo reaction) • Higher the activation energy, lower the rate • Transition state- Energy barrier has to be overcome

30. Reaction coordinate diagram

31. Mechanism of enzyme action • lower energy status of transition state is d/t – *Substrate strain *Proper orientation of substrates *Proximity of substrates *Change of electrostatic environment around the substrates

32. Michaelis- Menten Theory • Enzyme E combines with a single substrate S to form Enzyme-Substrate complex ES at the active site, which immediately dissociates to form free enzyme E and the product P S+ E ES E+P

33. Models to explain mechanismof specificity and catalysis • Formation of ES complex can be explained by 2 models: • Fischer’s Lock and Key Model • Koshland’s Induced Fit Model

34. Fischer’s Lock and Key Model

35. Fischer’s Lock and Key Model • Theory active site of enzyme is pre-shaped & rigid • Is complimentary to the substrate • Fit exactly into one another like key in lock • Explains only enzyme specificity • Fails to explain the flexibility shown by allosteric enzymes

36. Koshland’s Induced Fit Model

37. Koshland’s Induced Fit Model • Hand in glove Model -interaction of S & E induces a conformational change in E like glove when hand is introduced • Model: *active site is not rigid *binding of substrate induces conformational changes in the enzyme – leads to precise orientation of the catalytic groups catalysis • Explains both enzyme specificity and catalysis

38. Nomenclature of enzymes • Describe type of reaction & add suffix “-ase” • Ex: Dehydrogenases proteases isomerases • Modifiers: hormone sensitive lipase cysteine protease RNA polymerase III

39. Nomenclature of enzymes • International Union of Biochemists (IUB): unique name and 4 digit EC code number • Enzyme name has 2 parts: -Names indicating substrate(s) & cofactor -Type of reaction catalyzed ( ends in ‘ase’) • Additional information in parenthesis

40. Classification of enzymes • The International Union of Biochemistry and Molecular Biology (IUBMB) • 6 major classes of enzymes • Mnemonic: OTHLIL Oh Thank Heaven Learning Is Lively

41. Classification of enzymes • Oxidoreductases oxidation-reduction • Transferases transfer of group of atoms • Hydrolases hydrolysis • Lyases cleavage of bonds without hydrolysis • Isomerases rearrangement of atoms • Ligases joining of molecules (using ATP)

42. Oxidoreductases • Catalyzes oxidation of one substrate with simultaneous reduction of another substrate or coenzyme Alcohol dehydrogenase Alcohol Aldehyde NAD+ NADH +H+ Malate dehydrogenase Malate Oxaloacetate NAD+ NADH +H+

43. Oxidoreductases AH2 + B A + BH2

44. Transferases • These enzymes catalyze transfer of a group other than H such as- amino, phosphoryl, methyl, from one substrate to another A- X+ B A + B- X

45. Transferases • Transfer groups from one substrate to another • ExHexokinase Hexose Hexose-6-phosphate ATP ADP Alanine transaminase Pyruvate Alanine Glutamate α-ketoglutarate

46. Hydrolases • These enzymes catalyze cleavage of a molecule by addition of water (hydrolysis) • cleaves ester, peptide, glycosidic bonds A–B + H2O A-OH+ B–H

47. Hydrolases • Ex Lactase Sucrase Lactase Lactose + H2O Glucose + Galactose Sucrase Sucrose + H2O Glucose + Fructose

48. Lyases • break bonds by other than hydrolysis Fructose 1, 6 bis phosphate Aldolase Dihydroxyacetone Glyceraldehyde-3- phosphate phosphate Fumarase Malate Fumerate + H2O

49. Isomerases • These enzymes produce isomers of substrates. • Include racemases, epimerases and cis-trans isomerases. AA'

50. Isomerases Phosphohexose isomerase Glucose 6-P Fructose P Phosphotriose isomerase Glyceraldehyde 3-P Dihydroxyacetone 3-P