1. 1 Enzymes

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

3. What Are Enzymes?

4. 4 Biological CatalystBiological Catalyst ProteinsMost enzymes are Proteins Not permanentlyNot permanently changed in the process Not consumedNot consumed

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 phosphodiester bonds Zymogen Isozymes

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

8. The Active Site

9. major groupsPlasma 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 physiological role in the blood. occur in blood in very minute amounts).

10. Enzymes composed wholly of protein are known as simple enzymes ComplexComplex 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 bonds and 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: Reaction Rate Optimum pH 3 5 7 9 11 pH Narrow range of activity Most lose activity in low or high pH

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

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

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

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

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

43. Sulfa Drug Is Competitive Inhibitor -COOH H 2 N- -SONH 2 H 2 N- Pteridine Precursor Folic acid Tetrahydro- folic acid Sulfanilamide Sulfa drug (anti-inflammation) Para-aminobenzoic acid (PABA) Bacteria needs PABA for the biosynthesis of folic acid Sulfa drugs has similar structure with PABA, and inhibit bacteria growth. 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 AllostericAllosteric means (“occupy another space”, other than that of the substrate). Allosteric enzymesAllosteric 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.) usually contain multiple subunitsAllosteric enzymes usually contain multiple subunits. modifiers or modulatorsThe molecules regulating the allosteric enzymes are called effectors (modifiers or modulators).

47. non-covalentlyAllosteric effectors bind non-covalently at 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 catalytic activity of the enzyme or both.

51. Covalent Modification Addition or removal of phosphate groups from specific serine, threonine, tyrosine The phosphorylated form of the enzyme may be activated or deactivated according to the specific enzyme

53. Result of Regulation

54. Blood Sample

55. Plasma X Serum Physiological Lab.

56. Thank You

57. 57 Enzymes catalyzeAre specific for what they will catalyze ReusableAre Reusable

58. Enzymes as indicator tools In healthy individuals, levels of these enzymes are almost constant as it reflects a steady state elevated enzyme activity in plasma may indicate tissue damage which is accompanied by increased release of intracellular enzymes.

59. Enzyme Stabilizes Transition State S P ES ES T EP STST Reaction direction Energy change Energy required (no catalysis) Energy decreases (under catalysis) T = Transition state Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166