CRYOENZYMOLOGY OF PAPAIN - REACTION-MECHANISM WITH AN ESTER SUBSTRATE

The reaction between papain and Nα-carbobenzoxy-L-lysine methyl ester has been studied at subzero temperatures in fluid aqueous dimethyl sulfoxide. We have previously shown that this cryosolvent has no adverse effects on the catalytic or structural properties of papain (Fink, A. L., & Angelides, K. J. (1976) Biochemistry 15, 5287). Monitoring the reaction by absorbance spectroscopy revealed maximal changes in the vicinity of 276 nm. When the reaction was initiated by mixing enzyme and substrate at very low temperatures, a series of three reactions, prior to rate-limiting deacylation, was observed. Reaction 1 had a pseudo-first-order rate constant >1 s-1 at —65 °C and is interpreted to correspond to the binding of substrate. Reaction 2 was pH* dependent, with an estimated pK:* of ≤3.4 and Kobsdlim = 8 X 10-2 s-1 at —40 °C, and an energy of activation (Ea) of 7.4 kcal mol-1. (pH* and pK* refer to the apparent protonic activity and pK in the cryosolvent.) Reaction 2 is attributed to a repositioning of the active-site imidazole. Reaction 3 had Ea = 10.2 kcal mol-1 and was also pH* dependent, with pK* = 4.8 and Kobsdlim = 8.9 X 10-4 s-1 at -16.5 °C. Reaction 3 is ascribed to formation of the acylenzyme. If papain inactivated by alkylation with Nα-tosvl-l-lysine chloromethyl ketone was used in place of active papain, none of these reactions was observed. On the other hand if S-methylthio-Cys-25-papain was used, reactions 1 and 2 were still present but reaction 3 was absent. Reactions 1 and 2 could also be detected by changes in the fluorescence emission of the enzyme. Although the rate of reaction 2 was pH* independent above pH* 4.0, the magnitude of the corresponding decrease in fluorescence emission was pH* dependent with a pK* of 5.1 at —36 °C. Reactions 2 and 3 were absent with mercuripapain. A detailed mechanism involving movement of the active-site imidazole, which accounts for many of the apparently contradictory previously reported pK assignments, is proposed. A key feature of this mechanism is the existence of two conformational states of the enzyme, one with the imidazole of His-159 hydrogen bonded to Asn-175 (pK1m ̃ 4; pKsh ̃ 8), and the other involving the imidazole protonated, and electrostatically interacting with the carboxylate of Asp-158 and the thiolate of Cys-25 (pK1m ̃ 8; pKSH ̃ 4). © 1978, American Chemical Society. All rights reserved.