This document discusses factors that affect the rate of enzymatic reactions, including enzyme concentration, substrate concentration, pH, temperature, and inhibitors. It explains that reaction rate increases with increasing enzyme or substrate concentration until the enzyme reaches saturation. The Michaelis-Menten equation models this relationship between reaction rate and substrate concentration. Different types of inhibitors, such as competitive and noncompetitive inhibitors, affect the Michaelis constant Km and maximum reaction rate Vmax in distinct ways. Competitive inhibitors increase Km while leaving Vmax unchanged, whereas noncompetitive inhibitors decrease Vmax without changing Km.

1. ENZYMES: KINETICS (PART III)
 Factors affecting
velocity of enzymatic
reaction:
 Enzyme concentration
 Substrate concentration
 pH
 Temperature
 Inhibitors

3. Enzyme concentration
 The velocity of an
enzyme-catalyzed
reaction is directly
proportional to the
concentration of
enzyme.
 This the first order
 reaction
velocity
amount of enzyme

4. Substrate concentration:
 For a typical enzyme catalyzed reaction,
as substrate concentration is increased,
velocity increases until it reaches a
maximum value
 The plot is a rectangular hyperbola
 When further increases in substrate
concentration do not further increase
velocity of reaction, because enzyme to be
“saturated” by substrate

5. Hyperbola graph

6. MICHAELIS-MENTEN EQUATION
 Most enzymes show Michaelis-Menten
kinetics and plot of reaction velocity Vi
against [S] has a hyperbolic shape.
 The Michaelis-Menten equation illustrates
the relationship between initial reaction
velocity (Vi) and substrate concentration
[S].

7. Typical enzymatic catalysis reaction
E + S E-S E + P
k1
k2
KS =
[E] [S]
[ES]
=
k2
k1
Ks (constant of equation of E-S complex
formation) depends upon the chemical nature
of enzyme and substrate only.
k3
Km =
k2 + k3
k1
Michaelis-Menten
constant
where k1, k2,k3- velocity constants
for the respective reactions

8. Michaelis-Menten Equation
V =
k2 [E] [S]
Km + [S]
or, with [E] held constant,
V =
Vmax [S]
Km + [S]
where, Vmax = k2 [E]
Vmax is the maximum rate possible to achieve
with a given amount of enzyme.
The only way to increase Vmax is by increasing
the [E].
In the cell, this can be accomplished by
inducing the expression of the gene encoding
the enzyme.

9. Km
Km is the substrate concentration required to
produce half the maximum velocity and
determine the affinity of the enzume for its
substrate.
The Km value is an intrinsic property of the
enzyme-substrate system and cannot be altered
by changing [S] or [E].
When comparing two enzymes, the one with the
higher Km has a lower affinity for its substrate.

10. Blood Glucose
Velocity
of glycolysis
glucokinase
Km
Km
hexokinase
Hexokinase have more higher affinity to glucose than
glucokinase.
Hexokinase (brain, muscles) -has high activity in
hypoglycemic condition
Glucokinase (liver) -has high activity in hyperglucemic
condition

11. Bridge to Pharmacology
 Methanol poisoning is treated with ethanol
administration.
 Both are substrates for alcohol
dehydrogenase (ADH), with more higher
affinity to ethanol (lower Km). Hence ethanol
compared with methanol.
 This prevents conversion of methanol to
formaldehyde which is toxic and not
metabolized further.

12. ANALYSIS OF PLOT IN A,B,C
POINTS

13. Point A
 When [S] much less than Km [S] << Km
 The Vi is the first order: Vi is proportional to the
concentration of substrate.
Vi =
VMAX [S]
Km + [S]
is equal Km
=
VMAX
[S]
Km
= k [S]

14. Point B
 When [S] = Km
 The velocity is half the maximal velocity.
Vi =
VMAX [S]
=
VMAX
22 [S]

15. Point C
– When [S] much greater than Km
– [S]>> Km
 The velocity is zero order: the velocity independent of
substrate concentration.
Vi =
VMAX [S]
Km + [S]
is essentialy equal
to [S]
=
VMAX
[S] = Vmax
[S]
Vi = Vmax
OR

16. Lineweaver-Burk equation is a reciprocal
form of the Michaelis-Menten equation
V [S]
1
=
Km
Vmax
1
+
1
Vmax
Graph a stright line. The data are
represented by the portion of the graph to
the right of the y-axis, but the line is
extrapolated into the left quadrant to
determine its intercept with the x-axis
(value -1/Km). The intercept of the line with
y-axis gives the value(1/Vmax)
0
1
Km
1
Vmax
Slope =
Km
Vmax
1
Vi
1
[S]

17. Effect of pH

18. Optimum of pH for some enzymes
 1.5 – 2.5 - Pepsin
 6.9-7.0- Salivary amylase
 7.0- Catalase
 7.0 – 8.5 - Lipase of pancreatic juice

19. Effect of temperature

20. Inhibitors
Class of Inhibitor Km Vmax
Competitive Increase No effect
Noncompetitive No effect Decrease

21. Competitive inhibition: Substrate
analogs
 Have properties similar
to substrates of the
enzyme. They are
bound by enzyme, but
cannot be converted
further and therefore
reversibly block some
of the enzyme.

22. Substrate analogs
 A higher substrates concentration is needed
to achieve a half maximum rate, the Km
increases.
 High concentration of substrate displace the
inhibitor again.

23. Competitive inhibition: Analogs of
transition state also act competitively
 The statin drugs (lovastatin,
simvastatin) used to control
blood cholesterol levels,
competitively inhibit HMG
CoA reductase in cholesterol
synthesis.
 Methotrexate, an
antineoplastic drug,
competitevly inhibit DHF
reductase needed for purines
and deoxythymidine
synthesis
 And hence decrease DNA
replication.

24. Non-competitive inhibition
 Inhibitors interact with group that is important
for enzyme activity, but not affected binding of
the substrate.
 Km –unchanged, but the concentration of
functional enzymes decreases and Vmax
also decreases.

25. “Suicide substrate” (example non-
competitive inhibition)
 Substrate analog.
 They bind reversibly,
but then they form a
covalent bond with
the active center of
enzyme
 Eg. Action of
Penicillin

26. Penicillin as
“suicide substrates”
 The site of action of penicillin is
muramoylpentapeptide
carboxylase – enzyme that is
essential for formation of bacterial
cell walls.
 The penicillin resembles the
substrate of this enzyme (a
peptide with the C-terminal
sequence D-Ala-D-Ala) and
reversibly bound in the active
center. Beta-lactam ring binds with
Ser residue by stable covalent
bond, blocking the active center.
 In dividing bacteria, the loss of
enzyme activity leads to the
formation of unstable cell walls
and death.

27. Allosteric enzymes

28. Lineweaver-Burk Plot of Competitive
and Noncompetitive Inhibition