1. ENZYMES A protein with catalytic properties due to its power of specific activation

2. Chemical reactions  Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY  During this part of the reaction the molecules are said to be in a transition state.

3. Reaction pathway

4. Making reactions go faster  Increasing the temperature make molecules move faster  Biological systems are very sensitive to temperature changes.  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”

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

6. Enzyme structure  Enzymes are proteins  They have a globular shape  A complex 3-D structure Human pancreatic amylase © Dr. Anjuman BegumDr. Anjuman Begum

7. The active site  One part of an enzyme, the active site, is particularly important  The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily © H.PELLETIER, M.R.SAWAYA ProNuC Database ProNuC Database

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 Jmol from a RCSB PDB file © 2007 Steve Cook H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES2007 Steve Cook STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

9. 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

10. The Lock and Key Hypothesis  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

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

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

13. 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)

14. The Induced Fit Hypothesis  This explains the enzymes that can react with a range of substrates of similar types Hexokinase (a) without (b) with glucose substrate http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

15. Understanding K m The "kinetic activator constant"  K m is a constant  K m is a constant derived from rate constants  K m is an approximation of the dissociation constant of E from S  Small K m means tight binding; high K m means weak binding

16. Understanding V max The theoretical maximal velocity  V max is a constant  V max is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality  To reach V max would require that ALL enzyme molecules are tightly bound with substrate  V max is asymptotically approached as substrate is increased

17. The turnover number A measure of catalytic activity  k cat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate.  Values of k cat range from less than 1/sec to many millions per sec

18. Factors affecting Enzymes  substrate concentration  pH  temperature  inhibitors

19. Substrate concentration: Non- enzymatic reactions  The increase in velocity is proportional to the substrate concentration Reaction velocity Substrate concentration

20. Substrate concentration: Enzymatic reactions  Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied.  If you alter the concentration of the enzyme then V max will change too. Reaction velocity Substrate concentration V max

21. 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

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

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

24. 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

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

26. 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?

27. 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

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

29. 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

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

31. 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…

32. 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

33. 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

34. Enzymes and pH

35. Enzymes and inhibitors  Inhibitors are molecules that prevent enzymes from 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

36. Active site directed inhibition (Competitive)  Inhibitor resembles the substrate enough to bind to active site and so prevent the binding of the substrate: Substrate Inhibitor Enzyme

37. Active site directed inhibition (Competitive)  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

38. Enzymes and competitive 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

39. Enzymes and competitive 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

40. Enzymes and competitive 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

41. Non-active site directed inhibition (Non-competitive)  Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Inhibitor Enzyme

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

43.  Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Enzyme Activity is permanently lost

44. Enzymes and non-competitive 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

45. Applications of inhibitors  Negative feedback: end point or end product inhibition  Poisons snake bite, plant alkaloids and nerve gases.  Medicine antibiotics, sulphonamides, sedatives and stimulants