1. Factors Affecting Enzyme Activity

2. Enzymes are large globular proteins… They have a precise 3-D shape Some have quaternary structure The ‘active site’ (blue) represents a tiny part of the molecule RuBisCo

3. Amino Acid

4. A reminder about protein structure Amylase Protein structure is achieved by the precise folding of secondary structures to form a tertiary structure held together by a range of bond types between R- groups (or ‘side-chains’)

5. Some reaction kinetics…

7. The ‘Lock and Key’ analogy

9. Induced fit

10. Enzymes and temperature: a tale of two effects Temperature / o C Collision rate of enzymes and substrates Number of enzymes remaining undenatured Reaction rate / arbitrary units

11. Enzymes and temperature Temperature / o C Increasing kinetic energy increases successful collision rate Reaction rate / arbitrary units

12. Enzymes and temperature Temperature / o C Permanent disruption of tertiary structure leads to loss of active site shape, loss of binding efficiency and activity Reaction rate / arbitrary units

13. Enzymes and temperature Temperature / o C Optimum temperature Reaction rate / arbitrary units

14. Enzymes and pH The precise shape of an enzyme (and hence its active site) depends on the tertiary structure of the protein Tertiary structure is held together by weak bonds (including hydrogen bonds) between R-groups (or ‘side-chains’) Changing pH can cause these side chains to ionise resulting in the loss of H-bonding…

15. Enzymes and pH pH Reaction rate / arbitrary units Either side of the optimum pH, the gradual ionising of the side-chains (R-groups) results in loss of H- bonding, 3 o structure, active site shape loss of binding efficiency and eventually enzyme activity Optimum pH

16. Enzymes and pH pH Reaction rate / arbitrary units This loss of activity is only truly denaturation at extreme pH since between optimum and these extremes, the loss of activity is reversible Optimum pH

17. Enzymes and pH

18. Enzymes and [S] [S] Initial reaction rate / arbitrary units As soon as a reaction begins, [S] begins to fall and so it is important that initial reaction rates are measured

19. Enzymes and [S] [S] Initial reaction rate / arbitrary units

20. Enzymes and [S] [S] Initial reaction rate / arbitrary units Increasing [S] increases collision rate and increases reaction rate

21. Enzymes and [S] [S] Initial reaction rate / arbitrary units All active sites are occupied. Enzymes are working at maximum rate. All active sites are not occupied

22. Enzymes and [S] [S] Initial reaction rate / arbitrary units Maximum turnover number or V max has been reached

23. Enzymes and [enzyme] [Enzyme] Initial reaction rate / arbitrary units Can we explain this in terms of the proportions of active sites occupied? What factor is limiting here?

24. Enzymes and inhibitors Inhibitors are molecules that prevent enzymes reaching their maximum turnover numbers Some inhibitors compete with the substrate for the active site Some inhibitors affect the active site shape by binding to the enzyme elsewhere on the enzyme Active site directed inhibition Non-active site directed inhibition

25. Active site directed inhibition Inhibitor resembles the substrate enough to bind to active site and so prevent the binding of the substrate: Substrate Inhibitor Enzyme

26. Active site directed inhibition Inhibitor resembles the substrate enough to bind to active site and so prevent the binding of the substrate: Substrate Enzyme/Inhibitor complex Enzyme activity is lost

27. Enzymes and active site directed inhibition [S] Initial reaction rate / arbitrary units At low [S], the enzyme is more likely to bind to the inhibitor and so activity is markedly reduced Uninhibited Inhibited

28. Enzymes and active site directed inhibition [S] Initial reaction rate / arbitrary units As [S] rises, the enzyme is increasingly likely to bind to the substrate and so activity increases Uninhibited Inhibited

29. Enzymes and active site directed inhibition [S] Initial reaction rate / arbitrary units At high [S], the enzyme is very unlikely to bind to the inhibitor and so maximum turnover is achieved Uninhibited Inhibited

30. Non-active site directed inhibition Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Inhibitor Enzyme

31. Non-active site directed inhibition Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Enzyme Active site is changed irreversibility

32. Non-active site directed inhibition Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Enzyme Activity is permanently lost

33. Enzymes and non-active site directed inhibition [S] Initial reaction rate / arbitrary units Can we explain this graph in terms of limiting factors in the parts of the graph A and B? AB