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-fructofuranosidase:
Sucrose + H2O
glucose + fructose
When [S] [E] : the rate is zero order with respect to sucrose.
Initial rate no longer increases at S higher than S4
v= Vmax = k2 ET
v/Vmax = k2 ES / k2 ET
ES/ ET
v/Vmax = ES/ ET
v/Vmax = ES/ E + ES How to know ES?
ES = E * S / Ks
Ks is the dissociation constant of the first step in the enzymatic reaction
under the physiologically common conditions that substrate is in great excess over
Enzyme ([S] [E]).
ES = E * S /(k-1+k2)/k1
Therefore:
ES = E * S / KM
v/Vmax = S / Km + S
This expression, the Michaelis-Menten
equation, is the basic equation of
enzyme kinetic.
The maximal velocity of a reaction, Vmax occurs at high substrate concentrations when
the enzyme is saturated, that is, S>> Km, and ET is entirely in the ES form v= Vmax
when
Since Ks is the dissociation constant of the Michaelis complex, as Ks decreases, the enzymes affinity
for substrate increases. KM in therefore also a measure of the affinity of the enzyme for its substrate,
provided k2/k1 is small compared to Ks, that is k2 k-1 so that the ES
P reaction proceeds more
slowly than ES reverts to E + S
This quantity is also known as the turnover number of an enzyme because it is the number of
reaction processes (turnovers) that each active site catalyzes per unit time.
Enzymes
Substrate
Catalase
H2O2
Carbonic anhydrase
HCO3-
400,000
Acetylcholinesterase
Acetylcholine
140,000
b-Lactamase
Benzylpenicillin
Fumarase
Fumarate
ATP
40,000,000
2,000
800
0.4
O
R O
H3CCNCCOCH3
H H
kcat / Km
R=
Glycine
1.3 10-1
Norvaline
CH2CH2CH3
3.6 102
Norleucine
CH2CH2CH2CH3
3.0 103
Phenylalanine CH2
1.0 105
(M-1 s-1)
Adapted from Mathews et al (2000) Biochemistry (3e) p.379
S >> Km
vi=Vmax
Vmax= k2Et
v= Vmax = k2 ET
Vmax= 10 x 10 -6 M/seg Km=10 x10-5 M
Si en el ensayo se usaron 5mg/L de preparacin enzimtica, entonces:
Al iniciar:
t = 0, S = So
A cualquier tiempo:
T=t S=S
X = (So-S)/So
- dS/dt = vi = So dX/dt
S pequeas
S grandes
Enzyme Inhibition
Many substances alter the activity of an enzyme by reversibly combining with it in a way
what influence the binding of substrate and/or its turnover number. Substances that reduce
an enzymes activity in this way are known as inhibitors
Competitive Inhibition
A substance that competes directly with a normal substrate for an enzymes substratebinding site is known as a competitive inhibitor.
Enzyme Inhibition
Competitive Inhibition
This
is
the
Michaelis-Menten
equation that has been modified by a
factor, , which is defined as
Enzyme Inhibition
Competitive Inhibition
Recasting
A plot of this equation is linear and has a slope of KM/Vmax, a 1/[S] intercept
of -1/ KM, and a 1/v0 intercept of 1/ Vmax
Enzyme Inhibition
Uncompetitive Inhibition
In uncompetitive inhibition, the inhibitor binds directly to the enzyme-substrate complex
but not to the free enzyme
In this case, the inhibitor binding step has the dissociation constant
The uncompetitive inhibitor, which need not resemble the substrate, presumably distorts
the active site, thereby rendering the enzyme catalytically inactive.
Enzyme Inhibition
Uncompetitive Inhibition
The double-reciprocal plot consists of a family of parallel lines with slope KM/Vmax, 1/v0
intercepts of /Vmax and 1/[S] intercept of -/KM
Enzyme Inhibition
Mixed Inhibition (noncompetitive inhibition)
A mixed inhibitor binds to enzyme sites that participate in both substrate binding and
catalysis. The two dissociation constants for inhibitor binding
Double-reciprocal
plots
consist of lines that have
the slope KM/Vmax, with a
1/v0 intercept of /Vmax
and 1/[S] intercept of -/
KM
Acylation
O
CH3C
NO2
O
C
O-H
C
+ H2O
Nitrophenol
CH3COOH
Kinetics of reaction
OC
HO
Two-phase
reaction
Time (sec)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169
DESVIACIONES A M&M
dES/dt = 0
ES = k1 E*S / (k-1 + k2)
dES/dt = k2 ES k3 ES
dES/dt = 0
ES = k2 ES / k3
v = dP1/dt = k2 ES
v = dP2/dt = k3 ES
v = k3 k2 ES / k3
v = dP2/dt = k3 ES
Vmax = k3 ET
ET= E + ES + ES
Vmax = k3 (E + ES + ES)
Vmax = k3 (E + k1 E*S / (k-1 + k2) + k2 ES / k3)
Vmax = k3 (E + k1 E*S / (k-1 + k2) + k2 k1 E*S / (k-1 + k2) / k3 )
v / Vmax
=
k2 k1 E*S / (k-1 + k2)
k3 E + k1 k3 E*S / (k-1 + k2) + k2 k1 E*S / (k-1 + k2)
v / Vmax
v / Vmax
v / Vmax
Vmax = k3 ET
k2 k1 S
k3 (k-1 + k2) + k1 k3 S + k2 k1 S
k2 k1 S
k3 (k-1 + k2) + k1 S (k2 + k3)
k2 S / (k2 + k3)
k3 (k-1 + k2) / k1(k2 + k3) + S
k2 k3 ET S / (k2 + k3)
k3 (k-1 + k2) / k1 (k2 + k3) + S
Dividir entre:
k1 (k2 + k3)
K-2+
Vmax f = k2 k3 ET
k-2 + k3 +k2
Vmax r = k-1 k-2 ET
k-1 + k2 +k-2
Ks = k-1 k-2 + k-1 k3 + k2 k3
k1( k2 + k-2 + k3)
Kp
K-2+
Keq = Vmax f * Kp
Vmax r * Ks
v = Vmax f Kp S Vmax r Ks P
KsKp + KpS + KsP
v = Vmax f S P / Keq
Ks + S + (Ks/Kp) P