Chapter 8 (continued)

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Chapter 8 (continued) Enzymes

• You should read the entire chapter.
• For the midterm, you will not be tested on the
  following topics
• kinetic details of different types of enzyme
  inhibition (pp. 266-268)
• kinetic details of allosteric enzymes (pp. 280,
  middle of page, to 281, middle of page).
• Details of regulation by phosphorylation (pp.
  282, near bottom of page, to 286, near bottom of
  page).
• Hexokinase and Enolase--but these will be
  included later, when we study glycolysis

Chymotrypsin
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Enzyme Kinetics Michaelis-Menton Equation
VmaxS Vo ____________
KM S


KM S when Vo Vmax
_____
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1 KM 1 ______
_______ ______ Vo VmaxS
Vmax
Double-Reciprocal or Lineweaver-Burk Plot
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Vmax S Vo ____________
KM S
Vmax k2 Etotal This is true
only for this specific (rather simple) mechanism!
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kcat or Turnover Numbers
Vmax kcat Etotal
More general
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Catalytic efficiency, kcat/KmMeasures how the
enzyme performs when S is low The upper limit
for kcat/Km is the diffusion limit - the rate at
which E and S diffuse together
kcatEtotalS _________________ Km S
kcatEtotalS _________________ Km
Vo

when S is low
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Many enzymes catalyze reactions with two or more
substrates
a. Mechanisms involving ternary complex
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Steady-State Kinetics Can Help Determine Mechanism
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Inhibitor (I) binds only to E, not to ES
Inhibitor (I) binds only to ES, not to E. This
is a hypothetical case that has never been
documented for a real enzyme, but which makes a
useful contrast to competitive inhibition
Enzyme Inhibition
Inhibitor (I) binds either to E and ES or to ES
alone.
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Competitive Uncompetitive
Mixed Inhibition
Inhibition Inhibition
Kmchanges while Vmax does not
Km and Vmax both change
Km and Vmax both change
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1 KM 1 ______
_______ ______ Vo VmaxS
Vmax
Double-Reciprocal or Lineweaver-Burk Plot
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Irreversible Inhibition
(Irreversible)
diisopropylfluorophosphate
chymotrypsin
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Enzyme Activity is Affected by pH
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Structure of Chymotrypsin
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Pre-steady state evidence for an acyl enzyme
intermediate
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Regulatory Enzymes important in controlling
flux through metabolic pathways
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Example of an allosteric regulatory enzyme

• Two views of aspartate transcarbamoylase
• 12 subunits 6 are catalytic and six are
  regulatory
• Regulatory subunits are shown in red and yellow

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Regulation by Feedback Inhibition
Conversion of L-threonine to L-isoleucine
catalyzed by a sequence five enzymes,
E1-E5 L-isoleucine is an inhibitory allosteric
modulator of E1
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2. Regulation by covalent modification
Most common
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2. Regulation by Covalent Modification Proteoly
tic cleavage of zymogen