Enzymes

1. Enzymes 1

2. Objectives • What are enzymes ? • Properties of enzymes • Classification • Factors Affecting Enzyme Action • Enzyme Kinetics

3. What Are Enzymes?

4. BiologicalCatalyst Most enzymes are Proteins Not permanently changed in the process Not consumed 4

5. Differ from inorganic catalysts in: Being sensitive to changes in pH , temperature , loss of activity by time specificity

6. Definitions • Ribozymes : are RNAs with catalytic activity. • catalyze the cleavage & synthesis of phosphodiesterbonds • Zymogen • Isozymes

7. Isoenzymes: • enzymes that catalyze the same reaction but may have genetically determineddifference in the amino acid sequence • Thus may have difference in their physical properties

8. The Active Site

9. Plasma enzymes 2 major groups: • 1-Functional plasma enzymes • perform specific physiological function. • Examples: • Zymogens (inactive precursors) of enzymes involved in blood coagulation. • 2-Non-functional plasma enzymes • no physiologicalrole in the blood. • occur in blood in very minute amounts).

10. Enzymes composed wholly of protein are known as simple enzymes • Complex enzymes, which are composed of protein + small molecule holoenzymes. • Protein component apoenzyme, while the non-protein component coenzyme or cofactor

11. assistance

12. Prosthetic group describes a complex in which the small organic molecule is permanently bound to the apoenzyme by covalent bondsand cosubstrate occurs when it is transiently attached. • Coenzymes are often derivatives of vitamins (FAD,NAD, CoA)

13. Catalytic efficiency: Typically 100-1000 substrate molecules transformed to product / second ?(by enz.) Turn over number : is the number of substrate moles converted to product/enzyme mol /sec.

14. Location within the cell: • Mitochondria: TCA cycle Fatty oxidation Decarboxylation • Cytosol: Glycolysis HMP Fatty acid synthesis • Lysosomes: Degradation of macromolcule

15. Nomenclature of Enzymes& Classification

16. Recommended name uricase ,glucosidase

17. Systematic Name • Currently enzymes are grouped into six functional classes by the International Union of Biochemistry & Molecular Biology (I.U.B.M.B

20. Regulation: Rate of product formation responds to the needs of the cells

21. Factors Affecting Enzyme Action

22. Temperature Optimum temperature Reaction Rate Low High Temperature

23. Substrate Concentration Maximum activity Reaction Rate substrate concentration

24. pH: Narrow range of activity Most lose activity in low or high pH Reaction Rate Optimum pH 3 5 7 9 11 pH

26. Time • This is because by time the substrate decreases (↓v1 ) , • the product increases(↑v-1 ) besides • the enzyme decreases due to denaturation.

27. How do enzymes Work? Enzymes work by weakening bonds which lowers activation energy 27

28. Substrate Transition state Product If enzyme just binds substrate then there will be no further reaction X Enzyme not only recognizes substrate, but also induces the formation of transition state

29. Enzymes Without Enzyme With Enzyme Free Energy Free energy of activation Reactants Products Progress of the reaction 29

30. The active site consists of certain groups e.g.-SH, -OH, COO-, NH3 + & imidazole. Some groups are concerned with binding of the substrate & that determines the specificity of the enzyme.

31. Other groups are concerned with catalysis. • In some enzymes these groups can participate in general acid-base catalysis. In others, catalysis may involve transient formation of a covalent enzyme-substrate complex.

37. Inhibition of enzyme activity • Inhibitor :is any substance that can diminish the velocity of the enzyme catalyzed reaction. • Competitive • Non competitive: Heavy metal ions (e.g. mercury and lead) should generally be prevented from coming into contact with enzymes as they usually cause such irreversible inhibition by binding strongly to the amino acid backbone. • Uncompetitive

38. Reversible Inhibition:Competitive

39. Non Competitive Inhibition:

42. Enzyme Inhibition (Mechanism) E + S→ES→E + P + I ↓ EI E + S→ES→E + P + + II ↓ ↓ EI+S→EIS E + S→ES→E + P + I ↓ EIS ← ← ← ↑ ↑ ↑ ↑ Uncompetitive Competitive Non-competitive E Substrate E X Cartoon Guide Compete for active site Inhibitor Different site Equation and Description [I] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [I]. [I] binds to [ES] complex only, increasing [S] favors the inhibition by [I]. [I] binds to free [E] or [ES] complex; Increasing [S] can not overcome [I] inhibition. Juang RH (2004) BCbasics

43. Sulfa Drug Is Competitive Inhibitor -COOH H2N- -SONH2 H2N- Para-aminobenzoic acid (PABA) Bacteria needs PABA for the biosynthesis of folic acid Folic acid Tetrahydro- folic acid Pteridine Precursor Sulfa drugs has similar structure with PABA, and inhibit bacteria growth. Sulfanilamide Sulfa drug (anti-inflammation) Adapted from Bohinski (1987) Modern Concepts in Biochemistry (5e) p.197

44. Regulation of Enzyme Activity • Substrate availability • Allosteric Effectors • Covalent modification • Induction & repression

45. Regulation maybe short term regulation or long term regulation • The long term regulation includes enzyme synthesis & repression • The short term regulation includes allosteric regulation& covalent modification

46. Allosteric Effectors • Allosteric means (“occupy another space”, other than that of the substrate). • Allosteric enzymes are enzymes whose activity at the catalytic site may be modulated by the presence of allosteric effectors at an allosteric site (i.e. at a site other than the active site.) • Allosteric enzymes usually contain multiple subunits. • The molecules regulating the allosteric enzymes are called effectors (modifiers or modulators).

47. Allosteric effectors bind non-covalentlyat a site other than the active site. • Binding of allosteric effectors at the allosteric site induces conformational changes at the catalytic site& this can alter the affinity of the enzyme for its substrate or modify the maximal catalyticactivity of the enzyme or both.