Biosc 1000

Sample Problem Answers Lecture 9

1. All of the serine protease share the same catalytic triad of Ser-His-Asp, so what determines
their specificity?
Binding of a substrate and catalysis are distinct operations, so the amino acids involved in
catalyzing the reaction may have little to do with positioning the substrate(s) appropriately
for the reaction to occur. Thus, we can think of proximity and orientation effects on
reaction rate as a separate contributor to the rate enhancement. With improved sequencing
methods, irreversible competitive inhibitors, and site direct mutagenesis, we can now
explore the impact of changing amino acid residues in and around the active site map out
the residues involved in each of these processes.
2. What would be the consequence of mutating the Asp residue of a serine protease active site
to Glu?
Glutamic acid, although negatively charged like aspartic acid, is larger because of the 2nd
methylene group in the side chain. The added size would probably interfere with the
hydrogen bond interaction with histidine. I predict a drastic reduction in the rate of the
reaction.
3. How would a change in pH affect the activity of a serine protease?
Affect protonation state of catalytic triad. An enzyme using acid-base catalysis would have
narrow range pH over which it could show optimal activity
4. All of the amino acids that are likely to participate in acid-base catalysis possess a common
characteristic what is that?
All have ionizable side chains.
5. Enzyme deficiencies are frequently the root of inherited metabolic disease. Because an
enzyme deficiency causes the accumulation of upstream intermediates in a metabolic
pathway, it is possible to identify the missing enzyme and provide the missing products of
the pathway exogenously. But this doesnt necessarily prevent accumulation of upstream
intermediates, since the early part of the pathway is still functional. How could you use
what you know about allosteric regulation to reduce the amount of substrates funneled into a
blocked metabolic pathway?
You could use an allosteric inhibitor to block the committed step of the pathway.

Biosc 1000
6. Why are regulatory enzymes often the first enzyme in a multiple-reaction sequence?
Generally the committed step of a reaction sequence is the first step, thus, stopping the
pathway before it gets started allows the input substrates to be funneled off to other uses.
Occasionally, as we will see in glycolysis, the committed step occurs after the formation of
an intermediate that can serve as the input substrate for a number of reaction sequences.
7. How are allosteric enzymes different from other enzymes?
Allosteric enzymes usually have quarternary structure, they are generally committed steps in
the reaction pathway and have distinct regulatory and active sites which recognize and bind
different molecules
8. Think back to our discussion of the concerted and sequential binding models presented when
we discussed hemoglobin cooperativity. Which model best fits the allosteric regulation of
ATCase activity?
Within the textbook is an analysis suggesting that the Concerted model best describes the
allosteric behavior of ATCase, although the sequential model has been described as being
better able to account for negative cooperativity. As with hemoglobin, the binding model is
probably intermediate between the concerted and sequential models.
9. Because of the cooperative substrate binding behavior of allosteric enzymes, can we
consider KM as a measure of the enzymes affinity for its substrate?
No, in general the affinity of an allosteric enzyme for its substrate(s) is described by a K 0.5
or [S]0.5, which is simply the [S] at half maximal velocity. Since the KM is defined as the
ratio of rate constants describing the association and dissociation of substrate to a single
binding site, it does not accommodate the complexity of binding multiple substrates to
multiple subunits.