Last lecture: MM Kinetic Analysis, Enzyme

1
Last lecture MM Kinetic Analysis, Enzyme
Inhibition Today Enzyme
Inhibition Serine Proteases, Acid
Proteases
2
Analysis of Kinetic Data
Double Reciprocal Plot (Lineweaver-Burk)
3
7. Regulation of Enzyme Activity by Reversible
Inhibitor Binding Small molecule
inhibitors or activators Three types of
reversible inhibitors (a) Competitive
inhibitors resemble substrates, compete
for active
site (b) Non-competitive inhibitors inhibitors
bind to second
site and effect
conformational
change (c) Uncompetitive
inhibitors inhibitor binds to ES complex
not
free enzyme (E)
4
Competitive Inhibition
5
Effects of Competitive Inhibition on Enzyme
Kinetics
6
Non-competitive Inhibition
7
Kinetic Scheme Non-competitive Inhibition
8
Non-Competitive Inhibition
intrinsic affinity of substrate/product
unchanged
9
Uncompetitive Inhibition
10
Uncompetitive Inhibition
11
Uncompetitive Inhibition
uncompetitive inhibition
Compound RoundupTM N-phosphonomethylglycine Targ
et 3-phosphoshikimate 3-carboxyvinyltransferase
Compound Li Target myo-inositol
monophosphatase
12
Uncompetitive Inhibition
13
Summary
-observed kinetics diagnostic of mechanism -KM
can change, Vmax, or both
14
Enzyme Mechanisms -Serine Proteases -Acid
Proteases
15
Serine Proteases
Trypsin, Chymotrypsin, Elastase...
16
Catalytic Triad
17
(No Transcript)
18
Serine Proteases
Acyl-enzyme Intermediate
19
Burst Kinetics
A
t
20
Burst Kinetics
A
release of p-nitrophenolate
t
21
Burst Kinetics
hydrolysis of acyl-ester
release of p-nitrophenolate
22
Transition State Stabilization in Serine Proteases
23
Serine Protease - Specificity
24
Competitive Inhibition - Serine Proteases
25
(No Transcript)
26
source United Nations
27
Global HIV Pandemic
source WHO
28
(No Transcript)
29
(No Transcript)
30
sourceCenter for Disease Control, Atlanta, Ga
31
HIV Virus Structure
32
HIV Life Cycle
33
Reverse Transcriptase (RT) Inhibitors
34
Polypeptide precursor
35
(No Transcript)
36
HIV-1 Protease
37
Acid Protease Mechanism
38
(No Transcript)
39
HIV-Protease Substrate
40
Saquinavir
KD 0.4 nM
Roche
41
Crixivan
KD 0.3 nM
Merck
42
(No Transcript)