1. ENZYMES
A protein with catalytic properties due to its power of specific activation
© 2007 Paul Billiet ODWS

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.
© 2007 Paul Billiet ODWS

3. Reaction pathway
© 2007 Paul Billiet ODWS

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”
© 2007 Paul Billiet ODWS

5. An enzyme controlled pathway
Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.
© 2007 Paul Billiet ODWS

6. Enzyme structure Enzymes are proteins They have a globular shape
A complex 3-D structure
Human pancreatic amylase
© Dr. Anjuman Begum
© 2007 Paul Billiet ODWS

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
© 2007 Paul Billiet ODWS

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)
© 2007 Paul Billiet ODWS

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
© 2007 Paul Billiet ODWS

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
© 2007 Paul Billiet ODWS

11. The Lock and Key Hypothesis
Enzyme may be used again
Enzyme-substrate complex E S P
Reaction coordinate
© 2007 Paul Billiet ODWS

12. The Lock and Key Hypothesis
This explains enzyme specificity
This explains the loss of activity when enzymes denature
© 2007 Paul Billiet ODWS

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)
© 2007 Paul Billiet ODWS

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
© 2007 Paul Billiet ODWS

15. Factors affecting Enzymes
substrate concentration pH
temperature
inhibitors
© 2007 Paul Billiet ODWS

16. Substrate concentration: Non-enzymic reactions
Reaction velocity
Substrate concentration
The increase in velocity is proportional to the substrate concentration
© 2007 Paul Billiet ODWS

17. Substrate concentration: Enzymic 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.
© 2007 Paul Billiet ODWS

18. The effect of pH Optimum pH values Enzyme activity Trypsin Pepsin pH 13 5 7 9 11
© 2007 Paul Billiet ODWS

19. The effect of pH Extreme pH levels will produce denaturation
The structure of the enzyme is changed
The active site is distorted and the substrate molecules will no longer fit in it
At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur
This change in ionisation will affect the binding of the substrate with the active site.
© 2007 Paul Billiet ODWS

20. The effect of temperature
Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature.
For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature)
Enzyme-controlled reactions follow this rule as they are chemical reactions
BUT at high temperatures proteins denature
The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.
© 2007 Paul Billiet ODWS

21. The effect of temperature
Temperature / °C
Enzyme activity 10 20 30 40 50
Q10
Denaturation
© 2007 Paul Billiet ODWS

22. The effect of temperature
For most enzymes the optimum temperature is about 30°C
Many are a lot lower, cold water fish will die at 30°C because their enzymes denature
A few bacteria have enzymes that can withstand very high temperatures up to 100°C
Most enzymes however are fully denatured at 70°C
© 2007 Paul Billiet ODWS

23. Inhibitors
Inhibitors are chemicals that reduce the rate of enzymic reactions.
The are usually specific and they work at low concentrations.
They block the enzyme but they do not usually destroy it.
Many drugs and poisons are inhibitors of enzymes in the nervous system.
© 2007 Paul Billiet ODWS

24. The effect of enzyme inhibition
Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly.
Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase.
© 2007 Paul Billiet ODWS

25. The effect of enzyme inhibition
Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis.
There are two categories.
© 2007 Paul Billiet ODWS

26. The effect of enzyme inhibition
Competitive: These compete with the substrate molecules for the active site.
The inhibitor’s action is proportional to its concentration.
Resembles the substrate’s structure closely.
Enzyme inhibitor complex
Reversible reaction
E + I EI
© 2007 Paul Billiet ODWS

27. The effect of enzyme inhibition
Succinate
Fumarate + 2H++ 2e-
Succinate dehydrogenase
CH2COOH
CHCOOH
COOH
CH2
Malonate
© 2007 Paul Billiet ODWS

28. The effect of enzyme inhibition
Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site.
Examples
Cyanide combines with the Iron in the enzymes cytochrome oxidase.
Heavy metals, Ag or Hg, combine with –SH groups.
These can be removed by using a chelating agent such as EDTA.
© 2007 Paul Billiet ODWS

29. 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
© 2007 Paul Billiet ODWS

31. Classification of enzymes. Units of enzyme activity.31

32. Naming of Enzymes Common names
are formed by adding the suffix –ase to the name of substrate
Example: tyrosinase catalyzes oxidation of tyrosine; cellulase catalyzes the hydrolysis of cellulose
Common names don’t describe the chemistry of the reaction
Trivial names
Example: pepsin, catalase, trypsin.
Don’t give information about the substrate, product or chemistry of the reaction 32

33. Principle of the international classification
All enzymes are classified into six categories according to the type of reaction they catalyze
Each enzyme has an official international name ending in –ase
Each enzyme has classification number consisting of four digits: EC:
First digit refers to a class of enzyme, second -to a subclass, third – to a subsubclass, and fourth means the ordinal number of enzyme in subsubclass 33

34. The Six Classes of Enzymes
1. Oxidoreductases
Catalyze oxidation-reduction reactions
- oxidases peroxidases dehydrogenases 34

35. 2. Transferases
Catalyze group transfer reactions 35

36. 3. Hydrolases
Catalyze hydrolysis reactions where water is the acceptor of the transferred group
- esterases peptidases glycosidases 36

37. 4. Lyases
Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination 37

38. 5. Isomerases
Catalyze isomerization reactions 38

39. 6. Ligases (synthetases)
Catalyze ligation, or joining of two substrates
Require chemical energy (e.g. ATP) 39

41. ENZYME KINETICS
Why study enzyme kinetics?
a) the precise scheduling of reactions
in a cell is important to the cell and our
understanding of its workings
b) enzyme mechanisms, e.g., the
number of kinetic steps and the detailed
chemistry can be learned (enzymology).
c) understanding enzyme function
leads to better drugs.

42. Definition: rate of a reaction
For an enzyme acting on its substrate,
just as an ordinary chemical reaction,
the rate of the reaction depends on the
concentration of substrate, S.
A reaction leading to formation of
product is written:
S P
Rate = change in P /change in time or
rate = v = Δ[P]/Δt

43. For a chemical reaction (as contrasted
to an enzymatically catalyzed one),
the rate is proportional to reactant [S].
A rate constant, k,
can be defined:
rate = v = Δ[P]/Δt
= k [S]
rate
[S]

44. -
In contrast, found empirically for enzymes:
rate depends on [S] but
hyperbolic curve &
plateaus
rate also depends
on the enzyme
concentration
rate
[S]

45. or Interpretation Michaelis-Menten Model E + S  E●S  E + P
where E●S is enzyme-substrate complex,
i.e., an intermediate complex.
rate stops increasing or plateaus
because the complex E●S becomes filled
at high [S]

46. Assigning rate constants to MM model:
k k3
E + S  E●S  E+ P k1
From this kinetic scheme, a relationship
can be derived for the rate or velocity of
the reaction:
Michaelis-Menten Equation
Vmax[S]
[S] + Km
gives hyperbolic curve on next slide.
V =

47. Vmax Vmax is approached asymptotically Vmax/2 v or rate Km [S]
Vmax, the maximum rate (plateau) is
k3 x [total enzyme]
Km =(k2 +k3)/k1, almost a binding constant
Vmax
Vmax is
approached
asymptotically
Vmax/2
v or
rate Km
[S]
Unit of velocity is μmoles/min×mg protein

48. Km = [S], where the velocity v = Vmax /2,
is called the Michaelis constant.
Km is in units of concentration
Km good estimate
for the optimum
concentration
of substrate.
Vmax
Vmax/2 v Km
[S]

49. A plot of v = Vmax[S]
[S] + Km
is not accurate enough to derive good
Km & Vmax.
Computer analysis is done.
Reciprocal Plot
A double reciprocal plot or
Lineweaver-Burk plot is linear and more
eye-appealing for presentation.
mathematically = “linear transformation”

50. Result is:
1/v = Km/Vmax●1/[S] + 1/Vmax
Looks like the linear y = m●x + b
m = slope b = intercept on y
1/v = Km/Vmax●1/[S] + 1/Vmax
“x”
Notice also intercept on “x” is -1/Km

51. 1/v
x x x x x x x
slope =
Km/Vmax
intercept
= 1/Vmax
-1/Km
1/[S]

52. Competitive Inhibition
presented as double reciprocal plot
Model:
E + S  E●S  E + P + I 
E●I
I resembles S
I binds at active site reversibly
E●I cannot bind to S so no reaction
substrate
inhibitor

53. Competitive Inhibition
Vmax
No I +I
+more I Km
In competitive inhibition, can always
add enough [S] to overcome inhibition.
 same Vmax

54. Competitive inhibition
Double reciprocal plot
+ more I +I
Same 1/v intercept,
same Vmax
Different slopes,
competitive
Inhibition changes apparent Km
Note: inhibition line always above no inhibition.
1/Vmax
No I
1/[S]

55. Molecular interpretation
for competitive inhibition
competitive inhibitor binds to same site
as the substrate (competes).
its structure usually resembles
substrate or product.
While the inhibitor is bound, the enzyme
cannot bind substrate and no reaction
possible.
Many pharmaceutical agents are
competitive inhibitors so are many toxic
substances.

56. Blood pressure is regulated in kidney by renin,
example: captopril
Blood pressure is regulated in kidney by renin,
a specific proteolytic enzyme, which acts on
angiotensinogen, the precursor for the active
regulator.
renin
angiotensinogen angiotensin I
asp-arg-val-tyr-ile-his-pro-phe-his-leu
converting enzyme
angiotensin II the active factor O
peptide captopril HS-CH2-CH-C-N COOH
captopril is ACE inhibitor CH3 pro-like here
(angiotensin converting enzyme}

57. Finding useful inhibitors
Trial & error
Molecular
modeling
Testing enzyme
inhibition
Testing safety
Example:
HIV Protease is a dimer.
inhibitor is shown at active site.
interactions involve both subunits.

58. Noncompetitive Inhibition
E + S  E●S E + P
I I
 
E●I  E●S●l
substrate
inhibitor
E●I and E●S●I not productive, depletes
E and E●S

59. Noncompetitive
Inhibition
+ I
1/v
No I
1/[S]
different slopes, different 1/v intercepts.

60. Molecular Interpretation:
Inhibitor binds the enzyme somewhere
different from where the substrate binds.
So the inhibitor does not care whether
substrate is bound or not.
Inhibitor changes the conformation
of the enzyme at the active site so no
reaction is possible with inhibitor bound.
E●I and E●S●I not productive

61. Irreversible Inhibition
reactive compounds
combine covalently to enzyme so as
to permanently inactivate it
(previous examples are all reversible)
almost all are very toxic
most bind to a functional group in
active site of enzyme to block that site

62. Example 1:
diisopropyl fluorophosphate (DFP) binds
covalently to serine in serine proteases
& acetylcholinesterase - tool for biochemists
sarin is a deadly nerve gas  Paralysis O
Isopropyl-O-P-O-CH2- AChE
CH3

63. Example 2:
penicillin and related antibiotics bind
covalently to a peptidase involved in
cell wall synthesis in bacteria
Staphylococci, Streptococci sp. and others