Enzymes

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 ?G 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
  103-1017 times)

6

• An example
• CO2 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.

11
(No Transcript)
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 4.2.1.1 (this is the Enzyme Commission
  number).
•  

14
(No Transcript)
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.

17
(No Transcript)
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 pH2. Why?
•  

29

•  
• Many mammalian enzymes have an optimum pH 7.4
  but pepsin has an optimum of pH2. 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 VmaxS 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 VmaxS
• Km S
• 1 Km S
• Vo VmaxS

37

• Vo VmaxS
• Km S
• 1 Km S
• Vo VmaxS
• 1 Km S
• Vo VmaxS VmaxS

38

• Vo VmaxS
• Km S
• 1 Km S
• Vo VmaxS
• 1 Km S
• Vo VmaxS VmaxS
• 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. 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
47
(No Transcript)
48

• Note that 1 unchanged ? Vmax is unchanged
• Vmax
•  
• But
• -1 changed ? -1 ? 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. 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
• http//homepages.north.londonmet.ac.uk/baxc/ms100
  5n