Enzyme Catalysis

1. Enzyme Catalysis Prof. Dr. Supartono, M.S Postgraduate Semarang University State

2. Objective To understand how enzymes work at the molecular level. Ultimately requires total structure determination, but can learn much through biochemical analysis.

3. To Be Explained • Specificity • For specific substrates • Amino acids residues involved • Catalysis • Amino acids involved • Specific role(s) • Regulation Supartono

4. Enzymes • Enzymes are catalysts. They increase the speed of a chemical reaction without themselves undergoing any permanent chemical change. • Enzymes are neither used up in the reaction, nor do they appear as reaction products. Supartono

5. Discovery of Enzymes • 1825 Jon Jakob Berzelius discovered the catalytic effect of enzymes. • 1926 James Sumner isolated the first enzyme in pure form. • 1947 Northrup and Stanley together with Sumner were awarded the Nobel prize for the isolation of the enzyme pepsin. Berzelius Sumner Northrup Stanley Supartono

6. Enzyme Characteristics • High molecular weight proteins with masses ranging from 10,000 to as much as 2,000,000 grams per mole • Substrate specific catalysts • Highly efficien, increasing reaction rates by a factor as high as 108 Supartono

7. Enzyme Nomenclature • The earliest enzymes that were discovered have common names: i.e. Pepsin, Renin, Trypsin, Pancreatin • The enzyme name for most other enzymes ends in “ase” • The enzyme name indicates the substrate acted upon and the type of reaction that it catalyzes Supartono

8. Enzyme Names Examples of Enzyme Names • Glutamic Oxaloacetic Transaminase (GOT) • L-aspartate: 2-oxoglutarate aminotransferase. Enzyme names tend to be long and complicated. They are often abbreviated with acronyms Supartono

9. Enzyme Mechanisms Enzymes lower the activation energy for reactions and shorten the path from reactants to products Supartono

10. Enzyme Mechanisms The basic enzyme reaction can be represented as follows: E + S  ES  E + P Enzyme Substrate Enzyme substrate Enzyme Product(s) complex The enzyme binds with the substrate to form the Enzyme-Substrate Complex. Then the substrate is released as the product(s). Supartono

11. Enzyme Mechanisms Diagram of the action of the enzyme sucrase on sucrose. E+SES E+P Supartono

12. Enzyme Specificity • The action of an enzyme depends primarily on the tertiary and quaternary structure of the protein that constitutes the enzyme. • The part of the enzyme structure that acts on the substrate is called the active site. • The active siteis a groove or pocket in the enzyme structure where the substrate can bind. Supartono

13. Cofactors • Cofactors are other compounds or ions that enzymes require before their catalytic activity can occur. • The protein portion of the enzyme is referred to as the apoenzyme. • The enzyme plus the cofactor is known as a holoenzyme. Supartono

14. Cofactors Cofactorsmay be one of three types • Coenzyme:A non protein organic substance that is loosely attached to the enzyme • Prosthetic Group: A non protein organic substance that is firmly attached to the enzyme • Metal ion activators:K+, Fe2+, Fe3+, Cu2+, Co2+, Zn2+, Mn2+, Mg2+, Ca2+, or Mo2+, Supartono

15. Types of Cofactors • Enzymes have varying degrees of specificity. • One cofactor may serve many different enzymes. Supartono 15

16. Types of Cofactors • Enzymes have varying degrees of specificity. • One cofactor may serve many different enzymes. Supartono

17. Enzymes and Cofactors Supartono

18. Factors Affecting Enzyme Activity

19. Enzymes and Reaction Rates Factors that influence reaction rates of Enzyme catalyzed reactions include • Enzyme and substrate concentrations • Temperature • pH Supartono

20. Enzymes and Reaction Rates • Atlow concentrations, an increase in substrate concentration increases the rate because there are many active sites available to be occupied • At high substrateconcentrationsthe reaction rate levels off because most of the active sites are occupied Supartono

21. Substrate concentration • The maximum velocity of a reaction is reached when the active sites are almost continuously filled. • Increased substrate concentration after this point will not increase the rate.  • Vmaxis the maximum reaction rate Supartono

22. Substrate concentration • Vmax is the maximum reaction rate • The Michaelis-Menton constant , Kmis the substrate concentration when the rate is½ Vmax • Kmfor a particular enzyme with a particular substrate is always the same Supartono

23. Effect of Temperature • Higher temperature increases the number of effective collisions and therefore increases the rate of a reaction. • Above a certain temperature, the rate begins to decline because the enzyme protein begins to denature Supartono

24. Effect of pH • Each enzyme has an optimal pH at which it is most efficient • A change in pH can alter the ionization of the R groups of the amino acids. • When the charges on the amino acids change, hydrogen bonding within the protein molecule change and the molecule changes shape. • The new shape may not be effective. Pepsin is most efficient at pH2.5-3 while Trypsin is efficient at a much higher pH Supartono

25. pH Supartono

26. Effects of pH on Enzyme Activity • Binding of substrate to enzyme • Ionization state of “catalytic” amino acid residue side chains • Ionization of substrate • Variation in protein structure Supartono

27. Temperature Supartono

28. Inhibitors • Covalent • Reversible • Irreversible • Non-covalent: reversible Supartono

29. 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. Supartono

30. Active Site Binding and Catalysis

31. General Characteristics • Three dimensional entity • Occupies small part of enzyme volume • Substrates bound by multiple weak interactions • Clefts or crevices • Specificity depends on precise arrangement of atoms in active site Supartono

32. Models • Lock and Key Model: the active site exists “pre-formed” in the enzyme prior to interaction with the substrate. • Induced Fit Model: the enzyme undergoes a conformational change upon initial association with the substrate and this leads to formation of the active site. Supartono

33. Enzyme Mechanics • An enzyme-substrate complex forms when the enzyme’s active site binds with the substrate like a key fitting a lock.  • The shape of the enzyme must match the shape of the substrate. • Enzymes are therefore very specific; they will only function correctly if the shape of the substrate matches the active site. Supartono

34. Induced Fit Theory Supartono

35. Induced Fit Theory • The substrate molecule normally does not fit exactly in the active site. • This induces a change in the enzymes conformation (shape) to make a closer fit. • In reactions that involve breaking bonds, the inexact fit puts stress on certain bonds of the substrate. • This lowers the amount of energy needed to break them. Supartono

36. Induced Fit Theory • The enzyme does not actually form a chemical bond with the substrate. After the reaction, the products are released and the enzyme returns to its normal shape. • Because the enzyme does not form chemical bonds with the substrate, it remains unchanged. • The enzyme molecule can be reused repeatedly • Only a small amount of enzyme is needed Supartono 36

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

38. Binding or Positioning Site(Trypsin) Supartono

39. Binding or Positioning Site(Chymotrypsin) Supartono

40. Catalytic Site(e.g. Chymotrypsin) Supartono

41. Probing the Structure of the Active Site Model Substrates

42. Model Substrates(Chymotrypsin) Supartono

43. Peptide Chain? All Good Substrates! Supartono

44. a-amino group? Good Substrate! Supartono

45. Side Chain Substitutions Good Substrates t-butyl- Cyclohexyl Supartono

46. ConclusionBulky Hydrophobic Binding Site Supartono

47. Probing the Structure of the Active Site Competitive Inhibitors

48. Arginase Supartono

49. Good Competitive Inhibitors Supartono

50. Poor Competitive Inhibitors All Three Charged Groups are Important Supartono