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Bioreactors 1
Bioreactors 1
2
Robert S. Langer
Chemical Engineer, MIT
– substrates
– active site
substrates
(reactants)
enzyme
Substrates bind to an
enzyme at certain places called active
sites.
The lock-and-key model helps
illustrate how enzymes function.
Enzyme Unease
[From Biochemistry, 3/E by Stryer, copywrited 1988 by Lubert Stryer. Used with
permission of W.H. Freeman and Company.]
11
Enzymes
Enzymes provide a pathway for the substrate to
proceed at a faster rate. The substrate, S, reacts
to form a product P.
S Slow P
E•S
Fast
E + S ⎯⎯→ E • S
k1
E • S ⎯⎯→ E + S
k2
13
E • S + W ⎯⎯→ P + E
k3
Enzymes - Michaelis-Menten Kinetics
rP = k3 (E • S )(W )
rE •S = 0 = k1 (E )(S ) − k 2 (E • S ) − k3W (E • S )
k1 (E )(S )
(E • S ) =
k2 + k3W
Et = (E ) + (E • S )
(E ) = Et
k1S
1 +
14 k 2 + k3W
Enzymes - Michaelis-Menten Kinetics
Vmax
k cat
rP = k3 (E • S )(W ) =
k3W Et S kcat Et S
=
k 2 + k3W K + S
+S M
k1
KM
rP = k3 (E • S )(W ) =
Vmax S
Km + S
15
Enzymes - Michaelis-Menten Kinetics
Vmax=kcatEt
16
Enzymes - Michaelis-Menten Kinetics
Michaelis-Menten Equation
VmaxS
rP = −rS =
KM + S
(Michaelis-Menten plot)
Vmax VmaxS1/ 2
Vmax Solving: =
2 K M + S1/ 2
-rs KM=S1/2
therefore KM is the
concentration at which the rate
17 S1/2 CS is half the maximum rate.
Enzymes - Michaelis-Menten Kinetics
1 1 KM 1
Inverting yields: = +
− rS Vmax Vmax S
Lineweaver-Burk Plot
1/Vmax
18 1/S
Types of Enzyme Inhibition
Competitive
E + I I • E (inactive)
Uncompetitive
E • S + I I • E • S (inactive )
Non-competitive
E • S + I I • E • S (inactive )
I • E + S I • E • S (inactive )
19
Competitive Inhibition
20
Competitive Inhibition
E + S
⎯⎯→
k1
⎯⎯ E • S ⎯⎯→
k3
E+P
k2
E + I
⎯⎯→
k4
⎯⎯ E • I (inactive )
k5
1) Mechanisms:
E + S → E S E S → E + S
E S → P + E E + I → EI
EI → E + I
rP = k 3C ES
21
Competitive Inhibition
2) Rate Laws:
rES = 0 = k1CSC E − k 2 C ES − k 3C ES
k1CSCE CSCE
CES = =
k 2 + k3 Km
k 3 CS C E
rP =
Km
rIE = 0 = k 4 C I C E − k 5C IE
CI CE k5
CIE = KI =
22 KI k4
Competitive Inhibition
CEtot
C Etot = CE + C ES + CIE CE =
CS C I
1+ +
k 3C Etot CS Km KI
rP =
CI K m
K m + CS +
KI
Vmax CS
− rS =
CI
CS + K m 1 +
KI
1 1 km CI 1
= + 1 +
23 − rS Vmax Vmax K I CS
Competitive Inhibition
From before (no competition): 1 = 1 + K M 1
Increasing C
− rS Vmax Vmax CS
I
Competitive
1 No Inhibition Competitive
rS KM
slope = 1 1 K M CI 1
Vmax = + 1 +
− rS Vmax Vmax K I CS
1
Intercept = 1
Vmax
CS
25
Uncompetitive Inhibition
Inhibition only has affinity for enzyme-substrate complex
⎯⎯→
k1
E +S E • S ⎯⎯→
k3
P
⎯⎯
k2
⎯⎯→
k4
I + E •S I • E • S (inactive )
⎯⎯
k5
(E • S ) = k1 (E )(S ) = (E )(S )
k 2 + kcat KM
From (2)
(I • E • S ) = k4 (I )(E • S ) = (I )(E • S ) = (I )(E )(S )
k5 KI KI KM
k5
KI =
k4
kcat (E )(S )
rp = kcat (E • S ) =
27 KM
Uncompetitive Inhibition
Total enzyme
Et = (E ) + (E • S ) + (I • E • S )
= (E )1 +
( S ) (I )(S )
+
KM KI KM
kcat Et (S )
rp =
K M 1 +
( S ) (I )(S )
+
KM KI KM
Vmax (S )
− rS = rP =
(I )
K M + (S )1 +
28
KI
Uncompetitive Inhibition
1 (I )
K M + (S )1 +
1
=
− rS Vmax (S )
KI
1 K 1 1 (I )
= M + 1 +
− rS Vmax (S ) Vmax KI
29
Lineweaver-Burk Plot for uncompetitive inhibition
Non-competitive Inhibition
30
Non-competitive Inhibition
E+S E·S P+E
+I -I -I +I
(inactive)I.E + S I.E.S (inactive)
Increasing I
1
−
rS Vmax CS
No Inhibition − rS =
(k M + CS )1 + CI
Both slope and intercept kI
changes
1 1 C I k M 1 C I
= 1 + + 1 +
1 − rS Vmax k I Vmax CS k I
CS
31
Summary: Types of Enzyme Inhibition
32