Nov 20 Liliya A. Yatsunyk Ph.D. Northwestern University,

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Nov 20 Liliya A. Yatsunyk Ph.D. Northwestern
University, "Synthesis, Structure, and Magnetic
Spectroscopies of Non-Planar Hemes as Models of
the Cytochromes b Heme Centers" 400 SL 130 Nov
21 Melinda Kangala, Graduate Student, Western
Washington University (Chemistry). Thesis
Defense. 300 p.m. BI 212 Nov 28 Danielle Dube
Ph.D. Stanford University "Chemical Tools to
Target and Understand Glycosylation" 400 p.m.
SL 130
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Enolase
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Figure 13-6 Schematic representation of the
pyrimidine biosynthesis pathway.
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Figure 28-6 The biosynthetic origins of
pyrimidine ring atoms.
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Figure 28-1 The biosynthetic origins of purine
ring atoms.
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Figure 28-2 The metabolic pathway for the de novo
biosynthesis of IMP.
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Figure 28-7 Metabolic pathway for the de novo
synthesis of UMP.
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Figure 18-13 Control of glycogen metabolism
in muscle.
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Stryer Fig. 10.37
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Ch 14 Kinetics!
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Measuring Enzyme Activity
E S ? E P
Identify participants in the reaction.
How can we tell if an enzyme is working?
Need some sort of an assay e.g. Chymotrypsin
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Chymotrypsin is a serine protease which
cleaves on the carboxyl side of aromatic amino
acids.
Although its physiological purpose is as a
protease, it can also act rather nonspecifically
as an ESTERASE.
What is this?
CH3-(CO)-O-C6H4-NO2 ? Acetate pnitrophenol
(yellow)
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Stryer Fig. 8.11
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Stryer Fig. 8.12 Determining initial velocity
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What other factors can affect enzyme activity?
Is the product stable or transformed into
something else?
E
pH
Temperature
Salt concentration
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Figure 14-1 Plot of lnA versus time for a
first-order reaction.
A ? P lnA lnA0 - kt
This equation describes a line
y b mx
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Figure 14-4b Transition state diagrams. (b) For a
spontaneous reaction, that is, one in which the
free energy decreases.
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Figure 14-5 Transition state diagram for the
two-step overall reaction A I P.
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Figure 14-6 The effect of a catalyst on the
transition state diagram of a reaction.
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Figure 14-7 Progress curves for the components
of a simple MichaelisMenten reaction.
After the first few milliseconds of a reaction,
a steady state is attained.
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k2
k1
E S ?ES?EP
k-1
(k-2 is negligible until products start to build
up)
Steady state conditions--ES remains relatively
constant over the course of the rxn until S
starts runing out.
Vo k2ES
k1ES k-1ES k2ES (k-1 k2)ES
Define a new constant ES/ES (k-1 k2)/
k1 KM
KmES SE
KmES ETS-ESS
E ET -ES
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ES(KM S) ETS
(ETS
And ES v/k2
ES ------------------- KM S
k2ETS
Define Vmaxk2ET
v ------------------ KM S
vmaxS
Michaelis-Menton equation
v ----------------- KM S
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Figure 14-8 Plot of the initial velocity vo of a
simple MichaelisMenten reaction versus the
substrate concentration S.
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An enzyme obeys Michaelis-Menten kinetics
with Vmax 1.8 umol ml-1 s-1 at an enzyme
concentration of 15 umol ml-1. Calculate kcat
and KM for the enzyme. Is the value you obtain
for KM what you would expect given your data?
Why or why not? S uM vo (umol ml-1
s-1) 1600 1.39 800 1.13 400 0.83 200
0.54 100 0.32
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An enzyme obeys Michaelis-Menten kinetics
with Vmax 1.8 umol ml-1 s-1 at an enzyme
concentration of 15 umol ml-1. Calculate kcat
and KM for the enzyme. Is the value you obtain
for KM what you would expect given your data?
Why or why not? S mM vo (mmol ml-1
s-1) 1600 1.39 800 1.13 400 0.83 200 0.
54 100 0.32
Ans kcat 0.12 s-1 KM 470 mM
Yes.
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New Kinetic Parameter
Kcat Vmax ET
when Kcat ltltS
Turnover Number
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Previously defined Vo k2ES and ES
ES KM
Vo kcatES/KM
When SltltKM. E?ET
Vo kcatETS/KM
Kcat/KM rate constant for interaction of E and
S (turnover number) Can be used to measure an
enzymes preference for different substrates.
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What do these values tell you?
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Determining kcat and KM from intial rate data
o
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Vmax 150-160?? Km ???
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Lineweaver-Burk plot
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Vmax 150-160?? Km ???
Lineweaver- Burk plot
Vmax 164 mM/min Km 32.2 mM
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Figure 14-9 A double reciprocal (LineweaverBurk)
plot.
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Table 14-1 Values of KM, kcat, and kcat/KM for
Some Enzymes and Substrates.
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How does the substrate contact the enzyme?
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Figure 14-10 Cross section through the active
site of human superoxide dismutase (SOD).
Cu2 and R 143 form the binding site for O2-.
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