3. Enzyme concentration
īŽ The velocity of an
enzyme-catalyzed
reaction is directly
proportional to the
concentration of
enzyme.
īŽ This the first order
īŽ reaction
velocity
amount of enzyme
4. Substrate concentration:
īŽ For a typical enzyme catalyzed reaction,
as substrate concentration is increased,
velocity increases until it reaches a
maximum value
īŽ The plot is a rectangular hyperbola
īŽ When further increases in substrate
concentration do not further increase
velocity of reaction, because enzyme to be
âsaturatedâ by substrate
6. MICHAELIS-MENTEN EQUATION
īŽ Most enzymes show Michaelis-Menten
kinetics and plot of reaction velocity Vi
against [S] has a hyperbolic shape.
īŽ 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.
11. Bridge to Pharmacology
īŽ Methanol poisoning is treated with ethanol
administration.
īŽ Both are substrates for alcohol
dehydrogenase (ADH), with more higher
affinity to ethanol (lower Km). Hence ethanol
compared with methanol.
īŽ This prevents conversion of methanol to
formaldehyde which is toxic and not
metabolized further.
13. Point A
īŽ When [S] much less than Km [S] << Km
īŽ 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
īŽ When [S] = Km
īŽ The velocity is half the maximal velocity.
Vi =
VMAX [S]
=
VMAX
22 [S]
15. Point C
â When [S] much greater than Km
â [S]>> Km
īŽ 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]
21. Competitive inhibition: Substrate
analogs
īŽ 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
īŽ A higher substrates concentration is needed
to achieve a half maximum rate, the Km
increases.
īŽ High concentration of substrate displace the
inhibitor again.
23. Competitive inhibition: Analogs of
transition state also act competitively
īŽ The statin drugs (lovastatin,
simvastatin) used to control
blood cholesterol levels,
competitively inhibit HMG
CoA reductase in cholesterol
synthesis.
īŽ Methotrexate, an
antineoplastic drug,
competitevly inhibit DHF
reductase needed for purines
and deoxythymidine
synthesis
īŽ And hence decrease DNA
replication.
24. Non-competitive inhibition
īŽ Inhibitors interact with group that is important
for enzyme activity, but not affected binding of
the substrate.
īŽ Km âunchanged, but the concentration of
functional enzymes decreases and Vmax
also decreases.
25. âSuicide substrateâ (example non-
competitive inhibition)
īŽ Substrate analog.
īŽ They bind reversibly,
but then they form a
covalent bond with
the active center of
enzyme
īŽ Eg. Action of
Penicillin
26. Penicillin as
âsuicide substratesâ
īŽ The site of action of penicillin is
muramoylpentapeptide
carboxylase â enzyme that is
essential for formation of bacterial
cell walls.
īŽ 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.
īŽ In dividing bacteria, the loss of
enzyme activity leads to the
formation of unstable cell walls
and death.