Aula_3 bioquimica_2025_II..........................

Clase de Bioquímica de Enzimas

1. Prof. Fernando Gonzales
[email protected]
ENZYMES
Lecture
Biochemistry

2. Enzymes

3. Enzymes
Conceptsyoushouldknow:
•pH, buffers and amino acids
•Propertiesand structureof Proteins
•Basic conceptsof thermodynamics

4. 1. General Properties
3. Cofactor, Coenzyme and Prosthetic Group
5. Active Site
Enzymes
2. Classification
4. Mechanism of Enzyme Action
5. Formation of Enzyme-Substrate complex

5. - Catalyze chemical reactions in the cell making them
faster, enzymes are the main biological catalysts.
General Properties
Catalytic Power
Enzymes can accelerate reactions as much
as 10
16
over uncatalyzedrates!
Ureaseis a good example
Catalyzed rate: 3x10
4
mol/sec
Uncatalyzedrate: 3x10
-10
mol/sec
Ratio is 1x10
14
!
Enzymes

6. Molecules/sec
Without
catalysis
Reaction RateEnzyme
Catalyzed
reaction
1.7 milliseconds
0.2 milliseconds
1 millisecond
General Properties
Enzymes
- Catalyze chemical reactions in the cell making them
faster, enzymes are the main biological catalysts.
Pancreaticprotease,catalyzesa
peptidebondbreakattheC-termend
Isomerase,catalyzesconversionof
dihydroxyacetonephosphateand
glyceraldehyde3-phosphate
Interconversionbetweencarbon
dioxideandbicarbonate

7. Enzymesspeedup reactions
•Acid/base cataysis

8. https://www.youtube.com
/watch?v=yk14dOOvwMk

9. Enzymesspeedup reactions
•Acid/base cataysis
•Covalentcataysis
•Electrostaticcataysis
•Proximityand orientationeffects

10. - Biomolecules, they are usually specialized proteins.
General Properties
•Their catalytic activity depends on the integrity of
their native protein conformation
•If an enzyme is denatured or dissociated into its
subunits, catalytic activity is usually lost
•Primary, secondary, tertiary, and quaternary
structures of protein enzymes are essential to their
activity
Enzymes

11. General Properties
- Enzymes are able towork under physiological
conditions (pH, temperature, pressure).
Enzymes

12. •Enzymes selectively recognize proper substrates over
other molecules
•Specificity is controlled by structure -the unique fit of
substrate with enzyme controls the selectivity for
substrate and the product yield
General Properties
-Enzymes have a high degree of specificity.
Enzymes

13. General Properties
- Highcapacity for Regulation. Regulation of enzyme
activity is achieved in a variety of ways:
•Controlovertheamountofenzymeprotein
producedbythecell
•Rapid,reversibleinteractionsoftheenzymewith
metabolicinhibitorsandactivators.
Enzymes

14. Enzyme Nomenclature
•Urease= urea-hydrolyzing enzyme
•Phosphatase= enzymes hydrolyzing phosphoryl
groups
•Catalase= peroxide-decomposing enzyme
•Proteases= proteolytic enzymes of the digestive
tract, trypsin and pepsin

15. Classification
International Classification of Enzymes (IUBMB - International Union of
Biochemistry and Molecular Biology)

16. ATP: D-glucose-6-phosphotransferase
classification number E.C.2.7.1.2
E.C.2.7.1.1 (ATP:D-hexose-6-phosphotransferase)
Classification and Nomenclature

17. Cofactor, Coenzyme and Prosthetic Group
Cofactor

18. Cofactor, Coenzyme and Prosthetic Group
Cofactor

19. Cofactor
Cofactor, Coenzyme and Prosthetic Group

20. Coenzyme
Cofactor, Coenzyme and Prosthetic Group

21. Fe
2+
Heme
Myoglobin
Fe
2+
Cofactor, Coenzyme and Prosthetic Group
Prosthetic Group

22. A velocidade de reação aumenta, o
equilíbrio não é alterado
+
+Transition
state
G
+
+
S→P
Activation
Energy
G’
o
Free-energy
change
S P
Substrate Product
S
Ground state
Free

Energy (G)
P
Ground state
Reaction coordinate
Mechanism of Enzyme Action

23. The Energy “hill”
•A favorable equilibrium does not mean that the
S→P conversion will occur
•There is an energy barrier between S and P: the
energy required for alignment of reacting groups,
formation of transient unstable charges, bond
rearrangements, and other transformations
required for
•To undergo reaction, the molecules must
overcome this barrier and therefore must be
raised to a higher energy level.

24. •The rate of a reaction reflects this activation
energy: a higher activation energy corresponds to
a slower reaction.
•Reaction rates can be increased by raising the
temperature and/or pressure, to overcome the
energy barrier
•Activation energy can be lowered by adding a
catalyst. Catalysts enhance reaction rates by
lowering activation energies.
The Energy “hill”

25. A velocidade de reação aumenta, o
equilíbrio não é alterado
+
+Transition
state
G
+
+
S→P
Activation
Energy
G’
o
Free-energy
change
S P
Substrate Product
S
Free

Energy (G)
P
Reaction coordinate
Mechanism of Enzyme Action

26. + E
Gcat
Activation
Energy
+
+
+
+
ES EP
S P
Substrate Product
G
+
+
+
+
S→P
Free

Energy (G)
Transition
state
Activation
Energy
G’
o
Free-energy
change
Reaction coordinate
Mechanism of Enzyme Action
S
E+ P

27. E + S ES E + PEP
+ E
Gcat
Activation
Energy
+
+
+
+
ES EP
G
+
+
+
+
S→P
Free

Energy (G)
Transition
state
Activation
Energy
G’
o
Free-energy
change
Reaction coordinate
Mechanism of Enzyme Action
S
E+ P

28. Enzymes Affect Reaction Rates, Not Equilibria
E = Enzyme
S = substrate
P = Product
ES = Transient complex enzyme:substrate
EP = Transient complex enzyme:product

29. Enzyme
Substrate
Van der Waals interactions, Electrostatic interactions, hydrogen
bonds, hydrophobic interactions.
Enzyme Active Site

30. FormationoftheEScomplexis
thefirststepintheenzymatic
catalysis
Lock-and-key Model
Enzyme-Substrate Complex
Induced Fit Model

31. Substrate
(metal stick)
Transition state
(bent stick)
Products
(broken stick)
Enzyme-Substrate Complex

32. Enzyme complementary to substrate
Enzyme
Magnets
A few product
or
no products
Enzyme-Substrate Complex
Lock-
and-key
Model

33. Enzyme complementary to transition state
Induced Fit
Model
Binding between Enzyme and Substrate stabilizes at the transition state
Enzyme-Substrate Complex

34. Enzyme complementary to transition state
Induced Fit
Model
Binding between Enzyme and Substrate stabilizes at the transition state
Enzyme-Substrate Complex

35. A velocidade de reação aumenta, o
equilíbrio não é alterado
+
+Transition
state
G
+
+
S→P
Activation
Energy
G’
o
Free-energy
change
S P
Substrate Product
S
Ground state
Free

Energy (G)
P
Ground state
Reaction coordinate
Mechanism of Enzyme Action

36. Introduction to Kinetics

37. What is Enzyme Kinetics?
•Kineticsis the study of the rate at which
compounds react
•Rate of enzymatic reaction is affected by
–Enzyme
–Substrate
–Effectors
–Temperature

38. Why study enzyme kinetics?
•Quantitative description of biocatalysis
•Understand catalytic mechanism
•Find effective inhibitors
•Understand regulation of activity

39. ZeroOrder.Therateofazero-orderreactionis
independentoftheconcentrationofthereactant(s)
FirstOrder.Therateofafirst-orderreactionvaries
linearlyontheconcentrationofonereactant
SecondOrder.Therateofasecond-orderreaction
varieslinearlywiththesquareofconcentrationsof
onereactant
Reactionorder

40. First order reaction
For a first-order process: A P
The reaction velocity, v, is given by:v = = = k[A]
dP
dt
dA
dt
The reaction velocity for such a first-order reaction is proportional to the
concentration of A. Such a reaction is also called a unimolecular reaction.

41. Asecond-orderreactioncaninvolvethereactionoftwoidenticalor
differentsubstratemolecules:
A + A product(s) or A + B product(s)
The velocity of the reaction is then:
v = = - = k[A]
2
or v = - = - = k[A] [B]
dP
dtdt
dA dA
dt
dB
dt
Second order reaction

42. Progress of the triose phosphate isomerase reaction
Over time, the
concentration
of the substrate
decreases and
the
concentration
of the product
increases.
Glyceraldehyde -3 phosohate Dihydroxyacetone phosphate
Triose phosphate isomerase

43. Relationship between enzyme concentration and
reaction velocity
The more
enzymes
present, the
faster the
reaction.

44. How to Take Kinetic Measurements

45. V
[S]
Glucose Veloc
0.1 M 20
0.2 M 30
0.4 M 40
0.8 M 45
0.1 0.2 0.4 0.8

46. When the enzyme concentration is held constant, the reaction velocity
varies with the substrate concentration, but in a nonlinear fashion. As
small amounts of substrate are added to the enzyme preparation,
enzyme activity (measured as the reaction velocity) increases almost
linearly.
However, enzyme activity increases less dramatically as more substrate
is added. Finallya point is reached beyond which enzyme activity
appears to level off as it approaches a maximum value with increase in
substrate. This plateau is known as maximum velocity (V
max). This
behavior shows that at low substrate concentrations, the enzyme quickly
converts all the substrate to product, but as more substrate is added, the
enzyme becomes saturated with substrate resulting in formation of a
hyperbola in the graph of a plot of reaction velocity verses substrate
concentration.
The curve expressing this relationship has the same general shape for
most enzymes (it approaches a rectangular hyperbola).
Reaction velocity and substrate concentration

47. A plot of reaction velocity versus substrate concentration
Here varying amounts of
substrate are added to a
fixed amount of enzyme. The
reaction velocity is measured
for each substrate
concentration and plotted.
The resulting curve takes the
form of a hyperbola (a
mathematical function in
which the values initially
increase steeply but
eventually approach a
maximum level).

48. 1)Measurements made to measure initial velocity (v
o).
At v
overy little product formed. Therefore, the rate at
which E + P react to form ES is negligible and k
-2is 0.
Therefore
Initial Velocity Assumption
E + S ES E + P
k
1
k
-1
k
2
k
-2

49. Steady State Assumption
E + S ES E + P
k
1
k
-1
k
2
Steady state Assumption = [ES] is constant. The rate of ES
formation equals the rate of ES breakdown

50. E + S ES
k
1
Rate of ES formation
Rate = k
1 [E] [S]
E
S+S
E

51. ES E + P
k
2
E
S
ES E + S
k
-1
Rate of ES breakdown
Rate = [ES](k
2+ k
-1)
E
S+
k
-1
Rate =
Rate = Rate 1 + Rate 2
k
2 [ES]k
-1[ES]+
E+P
E
E
S
E

52. Therefore………if the rate of ES formation
equals the rate of ES breakdown
1) k
1[E][S] = [ES](k
-1+ k
2)
2) (k
-1+ k
2) / k
1 =[E][S] / [ES]
3) (k
-1+ k
2) / k
1 = K
m (Michaelisconstant)
Formation Rate = Breakdown Rate

53. E
E
E
E
E
E
E
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
SS
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
Kinetics Enzyme
V
0
Substrate Concentration [S]mM
Initial
Velocity
(
uM
/min)
E
E
E
E
E
E
E
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
SS
S
S
S
E
E
E
E
E
E
E
S
S
S
MAX
V
Saturation
V
max= velocity where all ofthe enzyme is bound to
substrate (enzyme is saturated with S)

54. Km
+ [S]
V
0
=
[S]MAX
V
k-1k2
Dissociation [ES]
k1 Formation [ES]
Km =
+
Michaelis-Menten Model
V
0
E+ S [E S]
k1
k-1
E+ P
k2
mM
(
uM
/min)
MAX
V
Km indicates affinity of an
enzyme for a substrate
Substrate Concentration [S]
Initial
Velocity

55. Effect of Substrate Concentration
•Ideal Rate:
•Deviations due to:
–Limitation of measurements
–Substrate inhibition
–Substrate prep contains inhibitors
–Enzyme prep contains inhibitorsSK
SV
v
m+
=
][
max

56. (
uM
/min)
Initial
Velocity
MAX
V
Km
+ [S]
V
0
=
[S]MAX
V
V
0
mMSubstrate Concentration [S]
Michaelis-Menten Model

57. MAX
V
Km
+ [S]
V
0
=
[S]MAX
V
MAX
V
2
2
V
0
=
MAX
V
Km
V
0
mM
K
m
= [S]
Substrate Concentration [S]
Michaelis-Menten Model

58. Km
+ [S]
V
0
=
[S]MAX
V
Km
V
0
=
[S]MAX
V
MAX
V
[S]
[S] <<< Km
V
0
E
E
E
E
E
E
E
S
S
S
mM
(
uM
/min)
Substrate Concentration [S]
Initial
Velocity
Michaelis-Menten Model

59. MAX
V
E
E
E
E
E
E
E
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
SS
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
SSaturation
Km
+ [S]
V
0
=
[S]MAX
V
V
0
=
MAX
V
[S]
[S] >>> Km
V
0
mM
(
uM
/min)
Substrate Concentration [S]
Initial
Velocity
Michaelis-Menten Model

60. What does K
mmean?
1.K
m= [S] at ½ V
max
2.K
mis a combination of
rate constants
describing the
formation and
breakdown of the ES
complex
3.K
mis usually a little
higher than the
physiological [S]

61. What does K
mmean?
4.K
mrepresents the amount of substrate required
to bind ½ of the available enzyme (binding
constant of the enzyme for substrate)
5.K
mcan be used to evaluate the specificity of an
enzyme for a substrate (if obeys Michaelis-
Menten)
6.Small K
mmeans tight binding; high K
mmeans
weak binding
Glucose Km = 8 X 10
-6
Allose Km = 8 X 10
-3
Mannose Km = 5 X 10
-6
Hexokinase
Glucose + ATP <-> Glucose-6-P + ADP

62. Limitations of M-M
1.Some enzyme catalyzed rxnsshow more complex behavior
2. E + S<->ES<->EZ<->EP<-> E + P
With M-M can look only at rate limiting step
2.Often more than one substrate
E+S
1<->ES
1+S
2<->ES
1S
2<->EP
1P
2<-> EP
2+P
1<-> E+P
2
Must optimize one substrate then calculate kinetic parameters for
the other
3.Assumes k
-2= 0
4.Assume steady state conditions

63. Case Study 1: Alcohol Dehydrogenase & ALDH2
Deficiency
• Enzyme Kinetics: ADH converts ethanol →
acetaldehyde, ALDH2converts acetaldehyde →
acetate
• Variant ALDH2*2 reduces Vmax drastically →
acetaldehyde accumulation
• Clinical: Facial flushing, tachycardia, nausea
after alcohol

64. Case Study 2: G6PD Deficiency and NADPH
Production
• Enzyme Kinetics: G6PDcatalyzes rate-limiting
step of pentose phosphate pathway
• Lower enzyme activity → reduced Vmax for
NADPH production
• Clinical: Oxidative stress in RBCs leads to
hemolysis (triggered by fava beans/drugs)

65. Case Study 3: Phenylketonuria (PKU) and Km
Variants
• Enzyme Kinetics: Phenylalanine hydroxylase
mutations alter Km for phenylalanine
• High Km = reduced affinity, enzyme less
effective at physiological [Phe]
• Clinical: Accumulation of phenylalanine →
neurotoxicity

66. A plot of reaction velocity versus substrate concentration
Here varying amounts of
substrate are added to a
fixed amount of enzyme. The
reaction velocity is measured
for each substrate
concentration and plotted.
The resulting curve takes the
form of a hyperbola (a
mathematical function in
which the values initially
increase steeply but
eventually approach a
maximum level).

67. ▪Vmax and Km are not likely to be determined by
increasing [S]
▪Insteadthe [S] vs. Vo data are transformed to a plot of
their reciprocal of each value.
▪1/[S] vs. 1/Vo
Lineweaver-Burk
(double reciprocal plot)

68. Km
+ [S]
V
0
=
[S]MAX
V
Lineweaver-Burk (double reciprocal plot)
Determination of Km and Vmax using Lineweaver-Burk equation
Km
[S]V
0
1
=
1
+
1
MAX
V
MAX
V
<
Km
+ [S]
V
0
[S]MAX
V
1
=
<
y=ax+b
slope

69. +
1
Vo
=
And this can be simplified to:
Vo =
V
max
[S]
[S] + K
m
Km + [S]
V
max
[S]
1
Vo
=(
Km
V
max
[S])
1 1
V
max
.
This is the equation for a straight line
Y = mX + b
Y = 1/Vo and X = 1 / [S]

70. Exercise
(mM)
1/[S] 1/V
2.54.54
1.49 3.44
1 3.125
0.5 2.5

71. 1/[S] 1/V
2.54.54
1.49 3.44
1 3.125
0.5 2.5
1/[S]
1/[V]
1 2
1
3
4
2.5
1/Vmax
-1/Km
2.3 = 1/Vmax
Vmax= 0.43
-2.31 = -1/Km
Km=0.43

73. Enzyme Inhibitors
Reversible versus Irreversible
•Reversible inhibition
1. Competitive inhibition
2. Uncompetitive inhibition
3. Mixed Inhibition
-Non-competitive inhibition
•Irreversible inhibitors

74. Reversible inhibition
•In competitive inhibition, the inhibitor is a substance that
directly competes with a substrate for binding to the
enzyme’s active site.
–Competitive inhibitors are molecules which are similar in shape
and chemical properties to substrates and capable of binding to
enzyme active sites. It lacks the exact electronic charge that
allows it to react
1. Competitive inhibition

75. Reversible inhibition
1. Competitive inhibition

76. Competitive
inhibitor
Reversible inhibition
1. Competitive inhibition

77. Reversible inhibition
1. Competitive inhibition

78. Reversible inhibition
1. Competitive inhibition

79. Reversible inhibition
1. Competitive inhibition

80. V
Km goes up → Affinity goes down
does not changeMax Becauseaddingmoresubstratecanovercome
theeffectottheCompetitiveInhibitor
→
Competitive
Inhibitor
Reversible inhibition
1. Competitive inhibition

81. Inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has
never been documented for a real enzyme, but which makes a useful
contrast to competitive inhibition
Apparent change in Vmax
Reversible inhibition
2. Uncompetitive inhibition

82. Reversible inhibition
2. Uncompetitive inhibition
Uncompetitive
Inhibitor
Vmax goes down

83. Reversible inhibition
2. Uncompetitive inhibition

84. •In mixed inhibition, the inhibitor binds to a site of
the enzyme other than the active site and elicits a
conformational change. As a result, the apparent
V
maxdecreases and the apparent K
mmay increase,
decrease or not change.
Reversible inhibition
3. Mixed inhibition

85. Reversible inhibition
3. Mixed inhibition

86. Km may increase, decrease or not change
Vmax decreases
Reversible inhibition
3. Mixed inhibition

87. Km increases
Vmax decreases
Km decreases
Vmax decreases
Km does not change
Vmax decreases
Non-competitive inhibition
When the K
m is not affected by the
inhibitor, it is said as non competitive
1
Vo
1
[S]
Mixed
Inhibitor
1
Vo
1
[S]
1
Vo
1
[S]