1. Enzyme Specificity Lecture 3

2. Objective To understand Specificity of enzymes

3. Specificity means: Ability of an enzyme to catalyse a specific reaction and NO others

4. The active site The active site, is a special region where catalysis occurs Occupies small part of enzyme volume The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily

5. Closer Look at Active site AS is not a point or line on enzyme It is a region in enzyme molecule where catalysis occurs AS has binding site and a catalytic site AS has a 3D Structure; residues widely separated in the primary structure are brought closer in the AS Clefts or crevices

6. AS: General Characterist ics Substrates bound by multiple weak interactions AS include both polar and nonpolar amino acids and create hydrophilic and hydrophobic microenvironment Specificity depends on precise arrangement of atoms in active site Substrates bound by multiple weak interactions Specificity depends on precise arrangement of atoms in active site

7. EnzymeAmino acid in active site HexokinaseHis PhosphoglucomutaseSer TrypsinSer, His Carbonic anhydraseCysteine CarboxypeptidaseHis, Arg, Tyr ThrombinSer, His AldolaseLys ChymotrypsinSer, his Choline esteraseSer

8. Cofactors An additional non-protein molecule that is needed by some enzymes to help the reaction Tightly bound cofactors are called prosthetic groups Cofactors that are bound and released easily are called coenzymes Many vitamins are coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

10. The Substrate The substrate of an enzyme are the reactants that are activated by the enzyme Enzymes are specific to their substrates The specificity is determined by the active site

12. Enzyme specificity can be for the type of reaction it catalyses or for its choice of substrates Substrate has a bond or linkage that can be attacked by the enzyme

13. Active site At the active site, functional groups of the enzyme interact with substrate (eg: RNase A)

14. This interaction may weaken one of its bonds or participate in a reaction by transforming an electron or proton (eg: catalytic mechanism of Serine proteases)

16. Enzyme and substrate binding releases ‘binding energy’. This free energy is used by enzyme to lower the activation energy of reaction and it provides specificity to reaction.

17. Chemical Reaction Pathway

18. Making reactions go faster Biological systems are very sensitive to temperature changes Increasing the temperature make molecules move faster Enzymes can increase the rate of reactions without increasing the temperature They do this by lowering the activation energy They create a new reaction pathway “a short cut”

19. An enzyme controlled pathway Most Enzyme controlled reactions proceed 10 8 to 10 11 times faster than corresponding non-enzymic reactions

20. Substrate binding site consists of an indentation or cleft on the surface of an enzyme. This cleft is complementary in shape to the substrate (geometric complementarity) The amino acid residues that form the binding site are arranged to interact specifically with the substrate in an attractive manner (electronic complementarity)

21. Two Models Lock and Key Induced Fit

22. The Lock and Key Hypothesis Proposed by Emil Fisher in 1894 the active site exists “pre-formed” in the enzyme prior to interaction with the substrate Fit between the substrate and the active site of the enzyme is exact, like a key fits into a lock very precisely The key is analogous to the enzyme and the substrate analogous to the lock Temporary structure called the enzyme-substrate complex formed Products have a different shape from the substrate Once formed, they are released from the active site, leaving it free to become attached to another substrate

23. The Lock and Key Hypothesis Enzyme may be used again Enzyme- substrate complex E S P E E P Reaction coordinate

24. The Lock and Key Hypothesis This explains enzyme specificity This explains the loss of activity when enzymes denature

25. The Induced Fit Hypothesis Some proteins can change their shape (conformation) When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation The active site is then moulded into a precise conformation Making the chemical environment suitable for the reaction The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

27. This model was proposed by Daniel Koshland in 1958 It requires the active site to be floppy and substrate to be rigid

28. Chemical Specificity 1. Group Specificity Enzymes may act on several different, though closely related substrates They catalyse reaction involving a particular group (eg: ALDH) ALDH catalyses the oxidation of variety of alcohols HK: assist the transfer of PO 4 from ATP to several different hexose sugars

29. 2. Absolute specificity Enzyme acting only on one particular substrate Eg: Glucokinase catalyses the transfer of PO 4 from ATP to glucose and to no other sugars (other egs: urease, arginase, catalase)

30. 3. Steriochemical specificity/ Geometric specificity Substrate chemically identical with different arrangement of atoms in in 3D space Only one of the isomers undergo reaction by a particular enzyme L-amino acid Ketoacid L-Amino acid Oxidase

31. Trypsin acts only on polypeptide containing L- amino acids, not those containing of D-amino acids Enzymes of glucose metabolism are specific for D-glucose

32. Yeast Alcohol Dehydrogenase (YADH) oxidises ethanol to aldehydes YADH acts on Methanol at 25 fold slower YADH acts on Propanol 2.5 fold slower NADPH (differ in a PO 4 at 2’ from NADH) does not bind to YADH

33. Glycerol Kinase phosphorylates glycerol to Glycerol-3-P If phosphorylation occurs at C1, the product is D- glycerol -3-P If phosphorylation occurs at C3, the product is L- glycerol -3-P The enzyme always produces only L-isomer Identical chemical groups in a substrate become different after binding at the microenvironment of the active site of the enzyme (1948, A.G. Ogston)

34. Enzyme Binding Sites Active Site = Binding Site + Catalytic Site Regulatory Site: a second binding site, occupation of which by an effector or regulatory molecule, can affect the active site and thus alter the efficiency of catalysis – improve or inhibit

35. Identification and Characterization of Active Site Structure: size, shape, charges, etc. Composition: identify amino acids involved in binding and catalysis.

36. Binding or Positioning Site (Trypsin)

37. Binding or Positioning Site (Chymotrypsin)

38. Catalytic Site (e.g. Chymotrypsin)

39. Probing the Structure of the Active Site Model Substrates

40. Model Substrates (Chymotrypsin)

41. Peptide Chain? All Good Substrates!

42.  -amino group? Good Substrate!

43. Side Chain Substitutions Good Substrates Cyclohexyl t-butyl-

44. Conclusion Bulky Hydrophobic Binding Site

45. Probing the Structure of the Active Site Competitive Inhibitors

46. Arginase

47. Good Competitive Inhibitors

48. Poor Competitive Inhibitors All Three Charged Groups are Important

49. Conclusion Active Site Structure of Arginase

50. Identifying Active Site Amino Acid Residues

51. Covalent Inactivation Diisopropyl Phosphofluoridate Inactivates Chymotrypsin by forming a 1:1 covalent adduct to Serine 195. Iodoacetic acid inactivates Ribonuclease by reacting with His 12 and His 119.

52. Affinity Labeling (General Approach) Positioning Group Reactive Group

53. Affinity Labeling (Tosyl- L -phenylalanine chloromethylketone) Inactivates Chymotrypsin by forming a 1:1 covalent adduct to Histidine 57

54. Trapping of Enzyme-Bound Intermediate (Chymotrypsin) Implicates Ser 195 in catalytic mechanism.

55. Mechanism