Chapter 14

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.

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?

As noted on page \(423,\) a true transition state can bind to an enzyme active site with a \(K_{\mathrm{T}}\) as low as \(7 \times 10^{-26} M .\) This is a remarkable number, with interesting consequences. Consider a hypothetical solution of an enzyme in equilibrium with a ligand that binds with a \(K_{\mathrm{D}}\) of \(10^{-27} M .\) If the concentration of free enzyme, \([\mathrm{E}],\) is equal to the concentration of the enzyme-ligand complex, [EL], what would \([\mathrm{L}],\) the concentration of free ligand, be? Calculate the volume of solution that would hold one molecule of free ligand at this concentration.

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?

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 \(37^{\circ} \mathrm{C}\)

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 \(37^{\circ} \mathrm{C}\)