A Structural Explanation for Enzyme Memory in Nonaqueous Solvents

Crystalline γ-chymotrypsin soaked in aqueous and nonaqueous solutions containing the inhibitor N-acetyl-D-tryptophan has been studied by X-ray crystallographic methods at a resolution of 2.2 Å. This inhibitor has been used to induce “enzyme memory” in chymotrypsin in nonaqueous solvents (Ståhl, M. et al. J. Am. Chem. Soc. 1991, 113, 9366–9368). The two soak solutions consisted of (a) the inhibitor, water, and 2-propanol and (b) the inhibitor, hexane, and 2-propanol. In the nonaqueous environment, the indole ring of the inhibitor binds in the specificity pocket of the enzyme for L-amino acids and not in a new site as had been suggested by others (Tawaki, S.; Klibanov, A. M. J. Am. Chem. Soc. 1992, 114, 1882—1884). No inhibitor binding was observed in the aqueous structure even though the concentration of inhibitor was identical to that in the nonaqueous experiment. Five hexane and three 2-propanol molecules were located in the nonaqueous structure. One of the three 2-propanol molecules occupies the P1' binding site. In the aqueous structure one 2-propanol was observed. In both structures, the addition of 2-propanol resulted in a dramatic increase in the number of observed water molecules. The differential binding affinity of N-acetyl-D-tryptophan in water and in a nonaqueous environment suggests a molecular explanation for the phenomenon of enzymatic memory.