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Last Lecture: Structure Determination, Heme Proteins

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2. Enzymes function under physiological conditions: T = ~40 C, pH ~ 7, p = 1 atm ... 4. Regulation: modulation of activity up and down, allosteric activation, ... – PowerPoint PPT presentation

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Title: Last Lecture: Structure Determination, Heme Proteins


1
Last Lecture Structure Determination, Heme
Proteins Today Enzymes
2
Enzymes
-most enzymes are polypeptides (important
exception - ribozymes!) -enzymatic activity
catalysis -catalysis (1) rate of
reaction increases (2) equilibrium
unchanged (3) catalyst (enzyme) not consumed
3
Ribozymes
Catalytic RNA
Nobel prize - Chemistry 1989
T.R. Cech (Colorado, HHMI)
S. Altman (Yale)
RNase P
Tetrahymena group I
http//almaz.com/nobel/chemistry/1989a.html
http//almaz.com/nobel/chemistry/1989b.html
4
Tetrahymena intron removal
Two sequential transesterifications
5 splice site
3
5
3
5
5
3
5
5
Contrasts with Simple Chemical Catalysis
1. Catalytic power 106-1012
ex. acetylcholine esterase
2. Enzymes function under physiological
conditions T 40C, pH 7, p 1 atm
6
Contrasts with Simple Chemical Catalysis
3. Specificity discrimination between various
substrates ex. amino-acyl tRNA
synthetases
4. Regulation modulation of activity up and
down, allosteric activation, product
inhibition
7
Mechanisms of Reaction
transition state (TS)
DG
8
Mechanisms of Reaction
9
Mechanisms of Enzymatic Catalysis
10
Mechanisms of Enzymatic Catalysis
-favourable stabilization of TS dipole, charge,
hydrophobic interaction
-not favourable, however enthalpic contributions
help pay the price, also positioning of
relevant functionalities
-acid/base catalysis, stabilization of developing
charge -change in mechanism from uncatalyzed
(including covalent catalysis)
11
Co-enzymes
vitamin B12
nicotinamide adenine dinucleotide (NAD)
12
Enzyme-Substrate Interactions
13
Enzyme-Substrate Interactions
14
Reaction Rates and Reaction Order
Zero Order Rate independent of reactant
concentration First Order Rate Equation Rate
of A B Second Order Rate Equation
2A B
15
First Order Reaction
Second Order Reaction
16
First Order Reaction
17
Second Order Reaction
18
Factors Affecting Rates of Enzyme Catalyzed
Reactions
1. Rate affected by Enzyme Concentration
formation of ES rate limiting
measure rate of appearance of product dP/dt
or disappearance of substrate
19
Factors Affecting Rates of Enzyme Catalyzed
Reactions
carry out reaction under saturating
concentrations of S ie SgtgtgtE rate is
independent of S, Rate k1E or Rate k2ES
20
Factors Affecting Rates of Enzyme Catalyzed
Reactions
2. Temperature rate of most chemical
reactions doubles with 10C increase in T
for many enzymes, triples
21
Factors Affecting Rates of Enzyme Catalyzed
Reactions
3. pH pH rate profile indicates proton
transfer in reaction
22
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Factors Affecting Rates of Enzyme Catalyzed
Reactions
4. Control of transcription, processing,
translation
25
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Factors Affecting Rates of Enzyme Catalyzed
Reactions
5. Limited Proteolysis
many enzymes biosynthesized as inactive
precursors (zymogens) that are activated by
limited proteolysis to yield mature active
enzyme ex. Chymotripsinogen, synthesizedin
pancreas, 245 aa
27
6. Substrate Concentration Effects
Michaelis-Menten
28
6. Substrate Concentration Effects
Assumptions 1. Initial rates, P 0
therefore no k-2 2. Steady State
(Briggs-Haldane) 3.
29
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33
The Michaelis Constant, KM
(3)
(2)
(1)
(1) (2) (3)
0 order reaction
34
Analysis of Kinetic Data
Double Reciprocal Plot (Lineweaver-Burk)
35
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)
36
Competitive Inhibition
37
Competitive Inhibition
38
Competitive Inhibition
39
Effects of Competitive Inhibition on Enzyme
Kinetics
40
Non-competitive Inhibition
41
Kinetic Scheme Non-competitive Inhibition
42
Non-Competitive Inhibition
intrinsic affinity of substrate/product
unchanged
43
Uncompetitive Inhibition
44
Uncompetitive Inhibition
45
Uncompetitive Inhibition
uncompetitive inhibition
Compound RoundupTM N-phosphonomethylglycine Targ
et 3-phosphoshikimate 3-carboxyvinyltransferase
Compound Li Target myo-inositol
monophosphatase
46
Uncompetitive Inhibition
47
Summary
-observed kinetics diagnostic of mechanism -KM
can change, Vmax, or both
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