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 108 to 1011 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 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

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.REES
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
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
Hexokinase (a) without (b) with glucose substrate
This explains the enzymes that can react with a range of substrates of similar types

15. Factors affecting Enzymes
substrate concentration pH
temperature
inhibitors

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

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

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

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

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

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

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

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

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

25. Enzymes and temperature
Increasing kinetic energy increases successful collision rate
Reaction rate / arbitrary units
Temperature / oC

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

27. Enzymes and temperature
Optimum temperature
Reaction rate / arbitrary units
Temperature / oC

28. 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…

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

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

31. Enzymes and pH

32. 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

33. 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

34. 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 activity is lost
Enzyme/Inhibitor complex

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

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

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

38. 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

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

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

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

42. 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

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

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

45. A measure of catalytic activity
The turnover number
A measure of catalytic activity
kcat, 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 kcat range from less than 1/sec to many millions per sec

46. Michaelis-Menten equation

47. Lineweaver-Burke plot

48. Hanes-Woolf plot