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Chapter 14: Problem 1

Tosyl-L-phenylalanine chloromethyl ketone (TPCK) specifically inhibits chymotrypsin by covalently labeling His \(^{57}\). Tosyl-L-phenylalanine chloromethyl ketone (TPCK) a. Propose a mechanism for the inactivation reaction, indicating the structure of the product(s). b. State why this inhibitor is specific for chymotrypsin. c. Propose a reagent based on the structure of TPCK that might be an effective inhibitor of trypsin.

Short Answer

Expert verified
Chymotrypsin and TPCK react in a nucleophilic acyl substitution reaction resulting in a covalent bond that inactivates the enzyme. TPCK is specific to chymotrypsin due to its ability to react specifically with the histidine residue and fitting into its hydrophobic pocket. An effective reagent for inhibiting trypsin might bear a structure similar to TPCK but with a positively charged group such as a guanidinium or amino group to fit into trypsin's binding pocket.

Step by step solution

01

Propose the mechanism for the inactivation reaction

Chymotrypsin and TPCK react in a nucleophilic acyl substitution reaction. The carbonyl carbon in the chloromethyl group of TPCK is attacked by the side chain nitrogen of His57 of chymotrypsin, forming a tetrahedral intermediate. This intermediate collapses expelling a chloride ion and leading to the formation of a covalent bond between TPCK and His57, thereby inactivating the enzyme.
02

Explain the specificity of TPCK for chymotrypsin

TPCK is specific to chymotrypsin due to the structural and reaction specificity. TPCK reacts specifically with the histidine residue in the catalytic triad of chymotrypsin and does not react with other enzymes that lack this critical active site histidine. Additionally, the bulky tosyl group on TPCK fits specifically into the large hydrophobic binding pocket of chymotrypsin.
03

Propose a reagent to inhibit trypsin

Trypsin has a different binding pocket preference than chymotrypsin, favoring positively charged arginine or lysine residues. Therefore, a potential inhibitor for trypsin would have a similar structure to TPCK but with a positively charged group instead of the tosyl group, which would fit into trypsin's binding pocket. A good candidate might be a compound with a guanidinium or amino group replacing the tosyl group in TPCK.

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Most popular questions from this chapter

An enzyme-substrate complex can form when the substrate \((\mathrm{s})\) bind (s) to the active site of the enzyme. Which environmental condition might alter the conformation of an enzyme to the extent that its substrate is unable to bind? a. Enzyme \(A\) at \(40^{\circ} \mathrm{C}\) b. Enzyme \(B\) at pH 2 c. Enzyme \(X\) at \(p H 4\) d. Enzyme \(Y\) at \(37^{\circ} \mathrm{C}\)

The \(k_{\text {cat }}\) for alkaline phosphatase-catalyzed hydrolysis of methylphosphate is approximately \(14 / \sec\) at \(\mathrm{pH} 8\) and \(25^{\circ} \mathrm{C}\). The rate constant for the uncatalyzed hydrolysis of methylphosphate under the same conditions is approximately \(10^{-15} /\) sec. What is the difference in the free energies of activation of these two reactions?

At \(35^{\circ} \mathrm{C},\) the rate of the reaction catalyzed by enzyme \(\mathrm{A}\) begins to level off. Which hypothesis best explains this observation? a. The temperature is too far below optimum. b. The enzyme has become saturated with substrate. c. Both \(A\) and \(B\). d. Neither A nor B.

In which of the following environmental conditions would digestive enzyme Y be unable to bring its substrate(s) to the transition state? a. At any temperature below optimum b. At any pH where the rate of reaction is not maximum c. At any pH lower than 5.5 d. At any temperature higher than \(37^{\circ} \mathrm{C}\)

Another consequence of tight binding (problem 9 ) is the free energy change for the binding process. Calculate \(\Delta G^{\circ}\) ' for an equilibrium with a \(K_{\mathrm{D}}\) of \(10^{-27} M .\) Compare this value to the free energies of the noncovalent and covalent bonds with which you are familiar. What are the implications of this number, in terms of the nature of the binding of a transition state to an enzyme active site?
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