This chapter covers the way how enzyme works and the enzyme kinetics

1. Chapter 3
Enzyme Mode of Action

2.  Enzyme molecules contain a special pocket or cleft
called the active sites.

3. Apoenzyme and Holoenzyme
 The enzyme without its non protein moiety is
termed as apoenzyme and it is inactive.
 Holoenzyme is an active enzyme with its non
protein component.

4. Cofactor
 A cofactor is a non-protein chemical
compound that is bound (either tightly or
loosely) to an enzyme and is required for
catalysis.
 Types of Cofactors:
 Coenzymes.
 Prosthetic groups.
Coenzyme: The non-protein component, loosely
bound to apoenzyme by non-covalent bond.
Examples : vitamins or compound derived from
vitamins.
Prosthetic group The non-protein component, tightly
bound to the apoenzyme by covalent bonds is called a
Prosthetic group.

5. Enzyme Specificity
 Enzymes have varying
degrees of specificity for
substrates
 Enzymes may recognize
and catalyze:
- a single substrate
- a group of similar
substrates
- a particular type of bond

15. Activation energy or Energy of Activation
 All chemical reactions require some amount of
energy to get them started.
OR
 It is First push to start reaction.
This energy is called activation energy.

16. Rate Enhancements

17. Examples of Enzymatic Activity.
Proteolytic Activity
A protease
Catalyzes cleavage of a peptide bond

18. Esterase Activity
Many proteases also manifest esterase activity
and catalyze cleavage of an ester bond.

19. Cleavage of a Peptide Bond
Enzymatic cleavage occurs on the
carboxyl side of the recognized sidechain.
Trypsin

20. 3.1. Mode of Action of Enzymes
• Enzymes increase reaction rates by
decreasing the Activation energy:
• Enzyme-Substrate Interactions:
‒ Formation of Enzyme substrate
complex by:
‒ Lock-and-Key Model
‒ Induced Fit Model

21. Lock-and-Key Model
 In the lock-and-key model of enzyme action:
- the active site has a rigid shape
- only substrates with the matching shape can fit
- the substrate is a key that fits the lock of the active
site
 This is an older model, however, and does not work
for all enzymes

22. Induced Fit Model
 In the induced-fit model of enzyme action:
- the active site is flexible, not rigid
- the shapes of the enzyme, active site, and substrate
adjust to maximumize the fit, which improves catalysis
- there is a greater range of substrate specificity
 This model is more consistent with a wider range of
enzymes

23. Enzymes
Lower a
Reaction’s
Activation
Energy

24. 3.2. The Science- How do Enzymes Work?
Enzymatic Catalysis
 Activation Energy (AE) –
The energy require to
reach transition state from
ground state.
 AE barrier must be
exceeded for rxn to
proceed.
 Lower AE barrier, the
more stable the transition
state (TS)
 The higher [TS], the move
likely the rxn will proceed.

25. Transition (TS) State Intermediate
 Transition state = unstable high-energy intermediate
 Rate of rxn depends on the frequency at which reactants
collide and form the TS
 Reactants must be in the correct orientation and collide
with sufficient energy to form TS
 Bonds are in the process of being formed and broken in
TS
 Short lived (10–14 to 10-13 secs)

26. Intermediates
• Intermediates are stable.
• In rxns w/ intermediates, 2 TS’s
are involved.
• The slowest step (rate
determining) has the highest
AE barrier.
• Formation of intermediate is
the slowest step.

27. •Enzyme binding of substrates decrease activation energy
by increasing the initial ground state (brings reactants
into correct orientation, decrease entropy)
•Need to stabilize TS to lower activation energy barrier.

28. ES complex must not be too stable
Raising the energy of
ES will increase the
catalyzed rate
•This is accomplished
by loss of entropy due
to formation of ES and
destabilization of ES
by
•strain
•distortion
•desolvation

29. Transition State Stabilization
Transition
state analog
• Equilibrium between ES <-> TS, enzyme drives
equilibrium towards TS
• Enzyme binds more tightly to TS than substrate

30. Mechanistic Strategies
Polar AA Residues in Active Sites

31. Common types of enzymatic mechanisms
 Substitutions rxns
 Bond cleavage rxns
 Redox rxns
 Acid base catalysis
 Covalent catalysis
 Metal Ion Catalysis

32. A) Substitution Rxns
 Nucleophillic Substitution–
 Direct Substitution
C
O
X
R
Y
C
O
X
R
Y
C
O
Y
R
+ X
C
R1 R2
R3 Y
C
X Y
R1 R2
R3
X
C
R1 R2
X R3
+ Y
transition state
Nucleophillic = e- rich
Electrophillic = e- poor

33. B) Oxidation reduction (Redox) Rxns
 Loose e- = oxidation (LEO)
 Gain e- = reduction (GER)
 Central to energy production
 If something oxidized something must be reduced
(reducing agent donates e- to oxidizing agent)
 Oxidations = removal of hydrogen or addition of
oxygen or removal of e-
 In biological systems reducing agent is usually a co-
factor (NADH of NADPH)

34. C) Cleavage Rxns
 Heterolytic vs homolytic cleavage
 Carbanion formation (retains both e-)
R3-C-H  R3-C:- + H+
 Carbocation formation (lose both e-)
R3-C-H  R3-C+ + H:-
 Free radical formation (lose single e-)
R1-O-O-R2  R1-O* + *O-R2
Hydride ion

35. D) Acid-Base Catalysis
 Accelerates rxn by catalytic transfer of a proton
 Involves AA residues that can accept a proton
 Can remove proton from –OH, -NH, -CH, or –XH
 Creates a strong nucleophillic reactant (i.e. X:-)
X H : B X: H B

36. X H : B X: H B
C
O
N
O
H H : B
C
O
OH
N
H
B
C
O
OH HN
: B
:
:
Acid-Base Catalysis
carbanion intermediate

37. E) Covalent Catalysis
 20% of all enzymes employ covalent catalysis
A-X + B + E <-> BX + E + A
 A group from a substrate binds covalently to
enzyme
(A-X + E <-> A + X-E)
 The intermediate enzyme substrate complex
(A-X) then donates the group (X) to a second
substrate (B)
(B + X-E <-> B-X + E)

38. Covalent Catalysis
Protein Kinases
ATP + E + Protein <-> ADP + E + Protein-P
1) A-P-P-P(ATP) + E-OH <-> A-P-P (ADP) + E-O-PO4
-
2) E-O-PO4
- + Protein-OH <-> E + Protein-O- PO4
-

39. F) Metal Ion Catalysis
Thermolysin is an endoprotease with a catalytic Zn2+ ion in the
active site. The Zn2+ ion stabilizes the buildup of negative charge
on the peptide carbonyl oxygen, as a glutamate residue
deprotonates water, promoting hydroxide attack on the carbonyl
carbon.

40. How Do Active-Site Residues Interact to Support
Catalysis?
 About half of the amino acids engage directly in
catalytic effects in enzyme active sites
 Other residues may function in secondary roles in
the active site:
 Raising or lowering catalytic residue pKa values
 Orientation of catalytic residues
 Charge stabilization
 Proton transfers via hydrogen tunneling

41. 3.3. Mechanism of Enzyme Action:
Enzyme Kinetics
Enzyme Kinetics
 Study of reaction rate and how they
changes in response to change in
experimental parameter is known as
kinetics
 Amount of substrate present is one of the
key factor affecting the rate of reaction
catalyzed by an enzyme in vitro

42. Low of Mass Action

43. Zero Order Reaction
 The rate is independent of the concentration of any
of the reactants

44. First Order Reaction

45. Second Order Reaction

47. Michaelis-Menten Kinetics
 The quantitative theory of enzyme kinetics was
proposed by two scientists Leonor Michaelis and
Maud Leonora Menten in 1913
 Enzyme reactions in Michaelis–Menten kinetics
theory occur in two stages:
1. The substrate binds reversibly to the enzyme,
forming the enzyme-substrate complex. This is
sometimes called the Michaelis-Menten complex
2. The enzyme then catalyzes the chemical step in
the reaction and releases the product

48. Enzyme Kinetics
E + S ES E + P
k1 k2
k-1
E = enzyme concentration
S = Substrate concentration
ES = Enzyme-substrate complex concentration
P = product concentration
k1 = rate constant for formation of ES from E + S k-
1 = rate constant for decomposition of ES to E + S
k2 = rate constant for decomposition of ES to E + P

49. Michaelis-Menton Plot
Graphical relationship between reaction velocity
and substrate concentration
E + S ES E + P
k1
k-1
k2
Zero order
(High [S])
1st
order
(low [S])

50. Finding Initial Veocity, Vo
Rate at the start of an enzyme catalyzed reaction

51. Enzyme Kinetics
•Enzyme-substrate complex (ES) - a non-covalent
complex formed when specific substrates fit into the
enzyme active site
When [S] >> [E], every enzyme binds a molecule of
substrate (enzyme is saturated with substrate)
Under these conditions the rate depends only upon
[E], and the reaction is pseudo-first order
E + S ES E + P
k1 k2
k-1

52. The Michaelis-Menton Equation
(1) Assume steady-state conditions:
Rate of ES formation = Rate of ES decomposition
(2) Define Michaelis constant: KM = (k-1 + k2) / k1
(3) The overall velocity of an enzyme-catalyzed
reaction is given by rate of conversion of ES to E + P.
vo = k2[ES] = kcat[ES]

53. Reaction Constituents
Time
ES
E
S
P

54. Michaelis-Menton Derivation
1. The overall rate of product formation: v = k2 [ES]
2. Rate of formation of [ES]: vf = k1[E][S]
3. Rate of decomposition of [ES]:
vd = k-1[ES] + k2 [ES]
4. Rate of ES formation = Rate of ES decomposition
(steady state)
5. So: k1[E][S] = k-1[ES] + k2 [ES]
E + S ES E + P
k1 k2
k-1

55. Michaelis-Menton Derivation
6. In solving for [ES], use the enzyme balance to
eliminate [E]. ET = [E] + [ES]
7. k1 (ET - [ES])[S] = k-1[ES] + k2 [ES]
k1 ET[S] - k1[ES][S] = k-1[ES] + k2 [ES]
8. Rearrange and combine [ES] terms:
k1 ET[S] = (k-1 + k2 + k1 [S])[ES]
k1 ET[S]
9. Solve for [ES] = -----------------------
(k-1 + k2 + k1 [S])

56. Michaelis-Menton Derivation
ET[S]
10. Divide through by k1: [ES] = -----------------------
(k-1 + k2)/k1 + [S]
11. Defined Michaelis constant: KM = (k-1 + k2) / k1
12. Substitute KM into the equation in step 10.
13. Then substitute [ES] into v = k2 [ES] from step1
and replace Vmax with k2 ET to give:
Vmax[S]
vo = -----------
KM + [S]

57. Michaelis-Menton Plot
Relates reaction velocity and substrate concentration
Vmax[S]
vo = -----------
KM + [S]

58. Lineweaver-Burke Plot
Also called a double reciprocal plot
1 KM 1 1
--- = ----- • ---- + -----
vo Vmax [S] Vmax

59. kcat
In an enzyme catalyzed reaction, the overall rate of
product formation is v = k2 [ES].
If all of the enzyme molecules are complexed with
substrate (excess [S]) then the maximum velocity
occurs and Vmax = kcatET where kcat is the overall
reaction rate constant.
This can also be written as kcat = Vmax /ET.
kcat is called the turnover number (TON).
E + S ES E + P
k1 k2
k-1

60. KM
• When KM = [S] when vo = 1/2 Vmax .
• KM @ k-1 / k1 = Ks (the enzyme-substrate dissociation
constant) when kcat is small (<< either k1 or k-1 ).
• Generally, the lower the numerical value of KM, the
tighter the substrate binding.
• KM is used as a measure of the affinity of E for S.

62. = kcat (s-1)

63. kcat/KM
kcat/KM is taken to be a measure of the efficiency
of an enzyme.
Rewriting kcat/KM in terms of the kinetic constants
gives:
kcat k1k2
---- = -----------
KM k-1 + k2
So, where k2 is small, the denominator becomes
k-1 and kcat/KM is small.

64. kcat/KM
kcat k1k2
---- = -----------
KM k-1 + k2
And where k2 is large, the denominator
becomes k2 and kcat/KM is limited by the value
of k1 or formation of the ES complex. This
formation is in turn limited by the rate of
diffusion of S into the active site of E. So, the
maximum value for this second-order rate
constant (kcat/KM) is the rate of diffusion (~109
sec-1 M-1).

65. Enzyme Inhibition
Noncovalent binding:
Competitive (I binds only to E)
Uncompetitive (I binds only to ES)
Noncompetitive (I binds to E or ES)
Covalent binding – irreversible
Group Specific
Substrate Analogs
Suicide

66. Competitive Inhibition

67. Competitive Inhibition
(I binds only to E)

69. Uncompetitive Inhibition

70. Uncompetitive Inhibition
(I binds only to ES)

71. Noncompetitive Inhibition

72. Noncompetitive Inhibition
(I binds to E or ES)

73. Irreversible - Group Specific

74. Irreversible - Group Specific

75. Irreversible - Substrate Analog

76. Irreversible - Substrate Analog

77. Irreversible - Suicide

79. Transition State Analog