1. Enzymes

2. GENERAL FEATURES
Enzymes are biological catalysts that speed up biochemical reactions.

3. GENERAL FEATURES
Enzymes are biological catalysts that speed up biochemical reactions.
They:
a) do not alter the ΔG of the reaction they catalyse but.....
b) they lower its activation energy and
c) .... enhance the rate of the reaction.

4. Nearly all enzymes are proteins although some RNA molecules have been shown to have catalytic activity.
Enzymes vary widely in size and structure.
They are (usually!) highly specific for the reaction that they catalyse.
The rates at which some enzymes operate are subject to regulation and control.

5. CATALYTIC POWER
This is the degree to which an enzyme speeds up a reaction.
The enzyme reaction rate can increase many times compared to the uncatalysed rate ( in the range times)

6. An example:
CO H2O H HCO3-
carbon dioxide CA bicarbonate ions
CA - carbonic anhydrase (present in red blood cells)
- increase reaction rate ~107 times
Without this catalytic power tissues would not be able to get rid of toxic CO2 rapidly enough.

7. SPECIFICITY
Each type of enzyme will only catalyse one particular chemical reaction (or set of closely related reactions)
Enzymes are specific with regard to substrate (reactants).
Note: this is generally true but some enzymes are able to accept a wide variety of substrates
eg cytochrome P450
This substrate is catalysed at the active site of the enzyme.

8. The Koshland induced fit model of enzyme catalysis

9. An example of specificity:

10. Product specificity:
With the same substrate, different enzymes catalyse the formation of different product.
Therefore different enzymes can effectively direct (or ‘funnel’) the same substance into different metabolic pathways.

12. NOMENCLATURE (or NAMING) OF ENZYMES
Enzymes are generally named after the specific reactions they catalyse.
Generally all should end in “-ase”, but (for historical reasons) there are some exceptions eg trypsin, papain, bromelain.
Enzymes can be defined unambiguously by their EC number (EC = Enzyme Commission). ~2000 have been classified in this way.
Many enzymes have common names as well as systematic names.

13. eg carbonic anhydrase (this is the common name)
= carbonate hydro-lyase (this is the systematic name)
= EC (this is the Enzyme Commission number).

15. ENZYME MEASUREMENT
How do we measure enzymes? In fact we measure their activity not their mass or concentration.
By carrying out an enzyme assay (assay means measurement) that measures the rate of the reaction catalysed.
To assay an enzyme:
take the enzyme (either pure or not),
add components needed for chemical reaction (substrates),
use appropriate conditions (pH, temperature, cofactors)
follow reaction over time (rate of reaction).

16. To calculate the rate of reaction plot a graph of the extent of reaction ( either formation of products or disappearance of reactants) against time.
This is called a Reaction-Time Plot or Progress Curve.

18. mmol
minutes

19. POINTS TO CONSIDER
How can you be sure that the reaction observed is enzyme catalysed ( and not just occurring at that rate anyway)?

20. POINTS TO CONSIDER
How can you be sure that the reaction observed is enzyme catalysed ( and not just occurring at that rate anyway)?
Ans. By including a control experiment - the enzyme blank. This contains everything except enzyme.
Usually these blanks show little or no measurable conversion of substrate.

21. Why does the enzyme-catalysed reaction ‘tail- off’ with time?

22. Why does the enzyme-catalysed reaction ‘tail- off’ with time?
Ans. For several possible reasons:
Enzyme becomes thermally inactivated with time
Product inhibition.
Substrate depletion.
Product build-up  reverse reaction.

23. Because of this ‘tailing-off’, the reaction rate varies.
The rate is actually measured in the early stages of the progress curve where the above factors have less influence.
This rate is called the initial rate or initial velocity (Vo) and is expressed as μmol substrate converted to product(s) per minute.

24. It is more useful to express enzyme rate not as initial rate but as specific activity.
Specific activity = μmol substrate converted per minute per mg protein.
= μmol min-1mg-1
Why?
Ans. Because it gives information on purity of enzyme. The higher the specific activity the greater the purity.

25. FACTORS AFFECTING ENZYME ACTIVITY
TEMPERATURE
For many reactions Q10 = 2, which means that for a 10 C rise in temperature the rate doubles.
This is true for enzyme reactions, except that if the temperature is held above the optimum temperature for too long a period then the enzyme inactivates.
NOTE: the optimum temperature varies according to species and time of exposure!

26. Enzymes become inactivated at higher temperatures because they are proteins
An enzyme may return to normal activity on cooling but only if it is able to renature.

27. pH
All enzymes are sensitive to pH, each enzyme having a particular optimum pH at which catalytic activity is maximum.
Enzymes are usually assayed (measured) at optimum pH. The optimum is maintained by using a buffered solution .

28. Many mammalian enzymes have an optimum pH = 7.4 but pepsin has an optimum of pH=2. Why?

29. Many mammalian enzymes have an optimum pH = 7.4 but pepsin has an optimum of pH=2. Why?
Ans. Because pepsin is a protease enzyme which operates in the stomach where the pH is low.

30. Proteins contain a large number of ionisable groups (due to side chains which carry a charge). The ionisation depends on the pKa of the ionisable group and the pH.
Altering the pH alters the degree of ionisation.
If one or more groups at the active site must be ionised to permit catalysis then altering the pH will affect the activity of the enzyme.

31. COFACTORS
Some enzymes contain one or more parts which are vital to enzyme activity. These are coenzymes, cofactors or prosthetic groups.
Their role is to aid catalysis. They often react with the substrates and they are located at the active sites of enzymes.

32. CONCENTRATION - RATE CURVES
If the initial rate is measured for progress curves at different [substrate] then a plot of concentration against rate can be obtained.
The Relationship between Rate of Reaction (Vo) and the
Substrate Concentration [S] for an Enzyme-Catalysed Reaction

33. The curve never reaches Vmax ( this is because a rectangular hyperbola is asymptotic!)
The concentration-rate curve is described by the following equation

34. Km is the Michaelis constant .
= a measure of affinity of that substrate for the enzyme - the lower the Km the greater the affinity
Because Vmax is never reached it can only be estimated from the concentration-rate curve
Vmax and Km are key paramemeters in defining how an enzyme functions.
How do we determine them?

35. Vo = Vmax[S] can be transformed by
Km + [S] taking reciprocals.
This allows us to plot the curve as a straight line in the form y = mx + c

36. Vo = Vmax[S]
Km + [S]
1 = Km + [S]
Vo Vmax[S]

37. Vo = Vmax[S]
Km + [S]
1 = Km + [S]
Vo Vmax[S]
1 = Km [S]
Vo Vmax[S] Vmax[S]

38. Vo = Vmax[S] Km + [S] 1 = Km + [S] Vo Vmax[S] 1 = Km + [S] Vo Vmax[S] Vmax[S] Plotting 1 against 1 gives a straight line. Vo [S]

39. A Lineweaver Burk plot

40. Note that it is not the only transformation possible
Note that it is not the only transformation possible. For example, the Hofstee-Eadie plot which arises from plotting Vo against Vo
[S]
Using these transformations , Km and Vmax can be determined easily and accurately.

41. INHIBITION
Two broad classes: irreversible and reversible.
Irreversible inhibition cannot be relieved by removing inhibitor - the enzyme is ‘poisoned’.
Reversible inhibition can be relieved by removal of the inhibitor from solution.   

42. Reversible inhibitors can be subdivided
a) competitive
b) non-competitive
c) uncompetitive

43. COMPETITIVE INHIBITION (CI)
Consider that there exists a molecule which resembles the substrate for an enzyme and the enzyme accepts it at the active site.
There are 2 possibilities:
i) the molecule is converted to a product - then it is a competing alternative substrate .
ii) the molecule does not undergo catalysis - then it is a competitive inhibitor.

44. Substrate
Competitive inhibitor

45. In other words a competitive inhibitor competes with the substrate for the active site.

46. In other words a competitive inhibitor competes with the substrate for the active site.
+CI Vo
[S]

48. Note that 1 unchanged  Vmax is unchanged
But:
-1 changed   Km changed
Km Kmapp

49. Km increases with increasing [Inhibitor] since more inhibitor occupies the active site effectively changing the affinity of the active site for the substrate.
As [S] increases Vo  Vmax since if [S] very large compared with [In], then S outcompetes In.

50. NON-COMPETITIVE INHIBITION (NCI)
Active site

51. NON-COMPETITIVE INHIBITION (NCI)

52. Note the differences between NCI and CI:
In NCI the Km remains the same remains the same in the presence of the inhibitor but the Vmax decreases.
The new Vmax is called Vmax apparent.

53. In UNCOMPETITIVE INHIBITION both the Km and Vmax are affected by the inhibitor.

54. Allosteric Enzymes
Allosteric enzymes have one or more sites in addition to the active site. These allosteric sites bind effector molecules which can modify the rate of catalysis.

55. This plot is S-shaped or sigmoidal and indicates
that the enzyme is allosteric.
V0 against [S]: Allosteric enzymes

56. -

57. Positive effectors or activators increase the rate of catalysis
Positive effectors or activators increase the rate of catalysis. Negative effectors or inhibitors decrease the rate of catalysis.
Allosteric enzymes are often multi-subunit proteins which catalyse the first (committed) reaction of a metabolic pathway.

58. MS1005N Web site: