1.  Strategic solutions. What are the four basic catalytic strategies used by many enzymes?

2.  Keeping busy. Many isolated enzymes, if incubated at 37°C, will be denatured. However, if the enzymes are incubated at 37°C in the presence of substrate, the enzymes are catalytically active. Explain this apparent paradox.

3.  Controlled paralysis. Succinylcholine is a fast-acting, short-duration muscle relaxant that is used when a tube is inserted into a patient’s trachea or when a bronchoscope is used to examine the trachea and bronchi for signs of cancer. Within seconds of the administration of succinylcholine, the patient experiences muscle paralysis and is placed on a respirator while the examination proceeds. Succinylcholine is a competitive inhibitor of acetylcholinesterase, a nervous system enzyme, and this inhibition causes paralysis. However, succinylcholine is hydrolyzed by blood-serum cholinesterase, which shows broader substrate specificity than does the nervous system enzyme. Paralysis lasts until the succinylcholine is hydrolyzed by the serum cholinesterase, usually several minutes later. ✓ 6

(a) As a safety measure, serum cholinesterase is measured before the examination takes place. Explain why this measurement is good idea.

(b) What would happen to the patient if the serum cholinesterase activity were only 10 units of activity per liter rather than the normal activity of about 80 units?

(c) Some patients have a mutant form of the serum cholinesterase that displays a K_M of 10 mM, rather than the normal 1.4 mM. What will be the effect of this mutation on the patient?

4.  Specific action. How can group-specific reagents be used to determine the mechanism of action of an enzyme? ✓ 6

5.  Like bread and butter. Match the term with the description or compound. ✓ 6

Competitive inhibition Competitive inhibition Competitive inhibition Uncompetitive inhibition Uncompetitive inhibition Uncompetitive inhibition Noncompetitive inhibition Noncompetitive inhibition Noncompetitive inhibition	Sulfanilamide Doxycycline Inhibitor binds at the active site Binds to the enzyme–substrate complex only Lowers Vmax and Roundup Inhibitor and substrate can bind simultaneously KM remains unchanged but Vmax is lower Vmax remains the same but the increases
Correct Matches:

8.  Informative inhibition. What are the four key types of irreversible inhibitors that can be used to study enzyme function? ✓ 6

9.  Self-preservation. Some bacteria produce the enzyme β-lactamase, which cleaves and opens lactam rings. How would the presence of β-lactamase affect bacterial sensitivity to penicillin? ✓ 6

10.  One for all and all for one. What is the catalytic triad, and what are the roles of the individual components in chymotrypsin activity?

11.  A burrow for oxyanions? What is the purpose of the oxyanion hole in chymotrypsin?

12.  Burst. What caused a “burst” of activity followed by a steady-state reaction when chymotrypsin was studied in the milliseconds subsequent to mixing the enzyme and substrate?

13.  Say no to cannibalism. If chymotrypsin is such an effective protease, why doesn’t it digest itself?

15.  Type 1 and type 2. In Section 7.2, we examined two types of bisubstrate reactions. Which of the two types best describes the action of chymotrypsin? Explain.

16.  Titration experiment. The effect of pH on the activity of an enzyme was examined. At its active site, the enzyme has an ionizable group that must be negatively charged in order for substrate binding and catalysis to take place. The ionizable group has a pK_a of 6.0. The substrate is positively charged throughout the pH range of the experiment. ✓ 6



(a) Draw the V₀-versus-pH curve when the substrate concentration is much greater than the K_M of the enzyme.

(b) Draw the V₀-versus-pH curve when the substrate concentration is much less than the K_M of the enzyme.

(c) At which pH will the velocity equal one-half of the maximal velocity attainable under the conditions described in (b)?

17.  Mutant. Predict the effect of mutating the aspartic acid at the active site of chymotrypsin to asparagine.

18.  No burst. Examination of the cleavage of the amide substrate A by chymotrypsin very early in the reaction reveals no burst. The reaction is monitored by noting the color produced by the release of the amino part of the substrate (highlighted in orange). Why is no burst observed?

19.  Variations on a theme. Recall that TPCK, an affinity label, inactivates chymotrypsin by covalently binding to histidine 57. Trypsin is a protease very similar to chymotrypsin, except that it hydrolyzes peptide bonds on the carboxyl side of lysine or arginine. ✓ 6

(a) Name an affinity-labeling agent for trypsin.

(b) How would you test the agent’s specificity?