SBS017 Basic Biochemistry Dr. Jim Sullivan j.a.sullivan@qmul.ac.uk

1. Lecture 9 Enzymes: Basic principles SBS017Basic Biochemistry Dr. Jim Sullivan j.a.sullivan@qmul.ac.uk

2. Learning Objectives Lecture 9 • You should be able to able to define the terms Enzyme, Specificity and Co-factor • You will understand the concept of Gibbs Free Energy and its relation to reaction equilibrium • You will be able to describe how enzymes effect the rate of biological reactions and be able to define the term Activation energy in the context of Transition state theory

3. Enzymes • Biological catalysts • Almost all enzymes are proteins (but RNA can have enzymic activity too, “ribozymes”) • Function by stabilizing transition states in reactions • Enzymes are highly specific

4. Enzymes accelerate biological reactions • e.g. Carbonic Anhydrase • CO2 + H2O H2CO3 • Each molecule of enzyme can hydrate 1,000,000 molecules of C02 per second, 10,000,000 times faster than uncatalysed reaction

5. Catalase • 2H2O2 2H2O+ O2

6. Enzymes are highly specific e.g Peptide bond hydrolysis (proteolysis) A. Trypsincleaves only after arginine and lysine residues. B.Thrombin cleaves between arginine and glycine only in particular sequences. But Papaincleaves all peptide bonds irrespective of sequence.

7. Specificity is important e.g. DNA polymerase I Adds nucleotides in sequence determined by template strand. Error rate of < 1 in 1,000,000 Due to precise 3D interaction of enzyme with substrate

8. Many enzymes require cofactors Cofactors are small molecules essential for enzyme catalysis Can be: • Coenzymes (small organic molecules) ii. Metal ions

9. Holoenzymes Enzyme without its cofactor “apoenzyme” With its cofactor “holoenzyme” Cofactors are essential for activity e.g. many vitamins are cofactors, many diseases associated with vitamin deficiency due to lack of specific enzyme activity

10. Energy transformation Many enzymes transform energy into different forms Adenosine Triphosphate (ATP) is universal currency Light ATP Photosynthesis Food ATP Respiration ATP work

11. ATP is an energy carrier e.g. ATP provides energy to pump Ca2+ across membranes

12. Free energy The free energy of a reaction is the difference between its reactants and its products This called the ΔG If ΔG is negative, the reaction will occur spontaneously “exergonic” If ΔG is positive, energy input is required “endogonic”:

13. Endogonic reactions Energy (G) Products ΔG positive Reactants Reaction coordinates

14. Exergonic reactions Energy (G) Reactants ΔG negative Products Reaction coordinates

15. Exergonic reactions Energy (G) H2O2 ΔG negative 2H2O + O2 Reaction coordinates

16. ΔG is independent of reaction path Energy (G) Reactants ΔG same Products Reaction coordinates

17. ΔG and reaction equilibria • A negative ΔG indicates that a reaction can occur spontaneously not that it will • ΔG tells us nothing about the rate of reaction only the energy of its end points • The rate of reaction is defined by the equilibrium constant

18. The equilibrium constant Keq A + B C + D

19. Calculating ΔG The free energy of a reaction is given by: • Where ΔG° is the standard free energy change (i.e. change at 1M concentrations), R is the universal gas constant and T the absolute temperature

20. Calculating ΔG at pH7 (ΔG’) At equilibrium and standard pH7:

21. Calculating ΔG at pH7 (ΔG’) Rearrange and substitute K’eq: At 25 °C, in log10, rearranged for K’eq: This means that a 10-fold change in Keq is equivalent to 5.69 kJ/mol difference in energy

22. Reaction equilibria Keq If [B] = 10 [A] = 1 Then ΔG = 5.69kJ/moll A B Energy (G) A 5.69 kJ/mol B Reaction coordinates

23. Reaction equilibria Keq If [B] = 100 [A] = 1 Then ΔG = 11.38 kJ/moll A B Energy (G) A 11.38 kJ/mol B Reaction coordinates

24. Catalysis • Enzymes cannot change equilibrium of reaction • ΔG is independent of reaction pathway • Enzymes accelerate rate of reaction Keq K+1 A B A B K-1

25. Catalysis No enzyme + enzyme K+1 A B K-1 Equilibrium is same but rate is 100x greater

26. Enzymes accelerate reaction rates Reaction reaches equilibrium much quicker with enzyme catalysis

27. Why do exergonic reactions (those with –ΔG) not take place spontaneously?

28. Activation energy • Reactions go via a high energy intermediate • This reduces the rate at which equilibrium is reached • The larger the activation energy the slower the rate

29. Transition state theory Transition state, S‡ Energy (G) ΔG‡ A ΔG B Reaction coordinates

30. Transition state theory • Enzymes reduces the activation barrier • Transition state energy becomes smaller

31. Transition state theory • The rate of reaction depends on ΔG‡ • Big ΔG‡ slow reaction • Small ΔG‡ faster reaction • Enzymes works by reducing ΔG‡ • Enzymes stabilise the transition state

32. Summary • Enzymes are biological catalysts • Enzymes are highly specific • Enzymes increase reaction rates but do not alter the equilibrium of reactions • Enzymes stabilise transition states, reducing activation energy and increasing the rate of reaction

33. Learning Objectives Lecture 9 • You should be able to able to define the terms Enzyme, Specificity and Co-factor • You will understand the concept of Gibbs Free Energy and its relation to reaction equilibrium • You will be able to describe how enzymes effect the rate of biological reactions and be able to define the term Activation energy in the context of Transition state theory

34. Lecture 10 Enzymes Kinetics SBS017Basic Biochemistry Dr. Jim Sullivan j.a.sullivan@qmul.ac.uk

35. Learning objectives Lecture 10 • You should be able to describe the evidence for enzyme/substrate complexes • You will be able to define the terms Active sites and Active site residues • You should be able to describe the Michaelis-Menten model for enzyme kinetics and the significance of KM and Vmax

36. Enzyme-Substrate (ES) complexes • Most enzymes are highly selective in binding of their substrates • Substrates bind to specific region of enzyme called Active Site • Catalytic specificity depends on binding specificity • Activity of many enzymes regulated at this stage

37. Evidence for ES complexes i. Saturation effect: At constant enzyme concentration reaction rate increases with substrate until Vmax is reached. Vmax is rate (or velocity) at which all enzyme active sites are filled.

38. Evidence for ES complexes ii. Structural data: Crystallography (3D structures), shows substrates bound to enzymes Cytochrome P450 (green) bound to its substrate camphor

39. Evidence for ES complexes iii. Spectroscopic data: Absorption and fluorescence of proteins and cofactors change when mixed together Changes in fluorescence intensity in the enzyme tryptophan synthetase after addition of substrates

40. Active sites • The active site of enzyme is region that binds the substrate • The catalytic groups are the amino acid side chains in the active site associated with the making and/or breaking of chemical bonds

41. Active sites i. The active site is a 3D structure formed by groups that can come from distant residues in the enzyme (tertiary structure) • 3D structure of Lysozyme, active site residues coloured • B. Linear schematic of Lysozyme amino acid primary sequence, active site residues coloured

42. Active sites ii. The active site takes up only a small volume of the enzyme iii. Active sites are unique chemical microenvironments, usually formed from cleft or crevice in enzyme.

43. Active sites Active sites often exclude water, and non-polar nature of site can enhance binding of substrates and allows polar catalytic groups to acquire special properties required for catalysis

44. Active sites iv. Active sites bind substrates with weak interactions Bonds can be electrostatic, hydrogen bonds, Van der Waals and hydrophobic interactions. ES complexes have equilibrium constants (Keq) on the 10-2to 10-8 range, equivalent to -3 to -12 kcalmol-1

45. Active sites v. The specificity of an enzyme for its substrate(s) is critically dependent on the arrangement of amino acid residues at the active site Remember importance of tertiary structure

46. Lock and key model • Proposed by Emil Fischer in 1890

47. Induced fit model • Substrates and enzymes are not rigid but dynamic and flexible • Daniel Koshland in 1958 proposed the induced fit model • In induced fit model the enzyme changes shape in order to optimise its fit to the substrate only after the substrate has bound

48. Induced fit model

49. Reaction kinetics First order reaction (uni-molecular): Rate of reaction is: Units of k: s-1 K A P

50. Reaction kinetics Second order reaction (bi-molecular): Rate of reaction is: Units of k: M-1s-1 K A + B P But if [A]>>[B] or [B]>>[A] reaction is Psuedo First Order