5/28/2014 3.

5 Solving Enzyme Kinetics Problems

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3.5 Solving Enzyme Kinetics Problems
1) Two strains of Bacterium sweetans, A and B, use sucrose (table sugar) as a sole carbon source. The first step
in the process of sucrose utilization is the passage of sucrose through a sucrose transporter protein in the
membrane. The characteristics of the two transport proteins are as follows (assuming [E]tot is the same in both):
Strain A B
KM 1000 mM 10 mM
Vmax 1000 mmol/min 100 mmol/min
a) Assuming that the rate of sucrose uptake is the rate limiting step in growth, which strain will grow faster
if the concentration of glucose is: 10 mM? 100 mM? 1000 mM?
b) One strain was isolated from the soil and the other from the floor at Toscanini's Ice Cream, which was
likely to be which? Why?
Solution:
Using the Michaelis-Menten equation, you can calculate the initial velocity of sucrose uptake for each strain
under the conditions listed. Note that this is the initial rate, as sucrose was depleted from the medium, the rate
would decrease. To simplify matters, assume that the number of cells is small so that the sucrose concentration
does not change appreciably during growth, so that the transport protein is always running at Vo.
For these calculations, substrate = sucrose.
Vo, enzyme of A: Vo, enzyme of B: Faster
[sucrose] Km=1000mM Km= 10mM Growing
mM Vmax=1000mmol/min Vmax= 100mmol/min strain:
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10 9.9 mmol/min 50 mmol/min B
100 91 mmol/min 91 mmol/min neither
1000 500 mmol/min 99 mmol/min A
b) It is likely that the [sucrose] levels in the soil are very low, so that strain B would be at a selective advantage
there. It is likely that a soil bacterium would have transport machinery adapted for low [sucrose].
It is likely that the [sucrose] on the floor at Toscanini's has a much higher [sucrose] than the soil. Under these
conditions, strain A would be at an advantage.
2) Given an enzyme with a Km = 10 mM and Vmax = 100 mmol/min.
a) If [S]=100mM, which will increase the velocity more: a 10-fold decrease in Km, or a 10-fold increase
in Vmax?
b) If [S]=10mM, which will increase the velocity more: a 10-fold decrease in Km, or a 10-fold increase in
Vmax?
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Solution:
You can answer these questions qualitatively or quantitatively. Having a qualitative understanding of what KM
and Vmax mean is more important than memorizing the Michaelis-Menten equation.
Qualitatively:
a) With [S]>>KM, the enzyme is running close to Vmax . Because of this, decreasing KM would hardly
increase V at all, but increasing Vmax 10-fold would increase V 10-fold.
b) With [S] = Km, the enzyme is running at 50% of Vmax. Decreasing Km 10-fold will raise V to near
Vmax, doubling V at most. Increasing Vmax 10-fold would also increase V 10-fold.
Quantitatively:
part Km Vmax [S] Vo result
-----------------------------------------------------------------------
a 10 mM 100 mmol/min 100 mM 91 mmol/min
a 1 mM 100 mmol/min 100 mM 99 mmol/min
a 10 mM 1000 mmol/min 100 mM 910 mmol/min greater effect
b 10 mM 100 mmol/min 10 mM 50 mmol/min
b 1 mM 100 mmol/min 10 mM 91 mmol/min
b 10 mM 1000 mmol/min 10 mM 500 mmol/min greater effect
3)The enzyme Carbonic Anhydrase (CA) catalyzes the following reaction:
CO2 + H20 <-----------> H2CO3
Given two identical sealed tubes with CO2 gas above buffer containing H2O and CO2 - one with added
carbonic anhydrase, one without. Allow the contents to come to equilibrium, then:
a) At equilibrium, which tube will have more CO2 in the gas phase, A or B?
b) Add CO2 to the gas phase of both tubes. Which tube will absorb it faster into the liquid, A or B?
Why?
c) Remove CO2 from the gas phase of both tubes. Which will replenish the CO2 faster, A or B? Why?
d) Take tube A, allow it to come to equilibrium, and inject a negligible volume of highly concentrated CA
into the liquid layer. Will CO2 gas be produced, absorbed, or will there be no change?
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e) Take tube B, allow it to come to equilibrium, and inject a negligible volume of highly concentrated ATP.
Will CO2 gas be produced, absorbed, or will there be no change?
Solution:
a) Since the tubes are at equilibrium, and since enzymes do not change the equilibrium concentrations of
reactants and products (another way of saying that they do not change Delta G), both tubes will have
identical [CO2] in the gas phase.
b) The CA in tube B will accelerate the absorption of CO2 by accelerating the conversion of CO2 into
H2CO3.
c) The CA in tube B will accelerate the production of CO2 by accelerating the conversion of H2CO3 into
CO2.
d) This is like part A, the CA cannot affect the equilibrium concentrations of reactants or products, only
alter the rate. So addition of CA to a tube at equilibrium will have no effect.
{However, addition of CA to a tube that was not at equilibrium could cause rapid absorption or
production of CO2. Normally, enzyme assays are carried out in reactions where the substrates have not
had enough time to reach equilibrium, so that the effect of adding enzyme will be detectable.}
e) Nothing will happen. Since ATP is not a substrate of CA, the concentrations of reactants and products
have not changed, so the equilibrium will not shift.
{If ATP were a substrate of CA, then adding it to the reaction would change the amount of CO2 in the
gas phase.}
4) The enzyme Lysozyme is present in human nasal mucus and catalyzes the breakdown of a component of the
cell wall of certain bacteria, causing them to burst. Lysozyme cleaves a polymer of N-acetyl-glucosamine
(NAG)n into smaller fragments:
lysozyme
NAG-NAG-NAG-NAG-NAG-NAG-NAG-NAG-NAG-NAG-NAG-NAG + H2O --------->
NAG-NAG-NAG-NAG-NAG-NAG + NAG-NAG-NAG-NAG-NAG-NAG
a) Given that the reaction proceeds as written, what can you conclude about the relative stability of
(NAG)n?
b) Given the following information, what can you conclude about the binding of lysozyme to its substrate?
Substrate Relative rate of hydrolysis
(NAG)2 0
(NAG)3 1
(NAG)4 8
(NAG)5 4,000
(NAG)6 30,000
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(NAG)8 30,000
c) If you treated a solution of (NAG)100 with lysozyme, what would the majority of the product be?
Solution:
a) Since lysozyme cannot effect the Delta G of the reaction, (NAG)n must be unstable (that is, the
reaction where it breaks into monomers has a Delta G<0).
{But why then do bacteria use an unstable molecule as a cell wall? Because, even though the (NAG)n will
eventually come to equilibrium and decompose into monomers, the uncatalyzed rate of hydrolysis is very
low, so reaching equilibrium might take decades. Lysozyme increases this rate enormously, and acts as a
bacteriocide.}
b) Even though the reaction catalyzed by lysozyme appears to be:
NAG-NAG + H2O --------> NAG + NAG
the substrate binding site must have a pocket designed to hold 5 or 6 NAG units before the enzyme can
get into the active conformation. This is an example of substrate specificity.
c) After a short time, it would mostly be (NAG)4, since any larger fragments will be rapidly cleaved. After
a long time, it will be (NAG)2 and (NAG)3.

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