P K perturbations of nitrophenol-containing reporter groups at the active site of papain

Abstract

The p K perturbations exhibited by nitrophenol reporter groups linked to the active site thiol group in papain were compared in order to probe the pH dependence of the charge in the environment of the active site of this enzyme. Nitrophenol reporter groups were introduced at the active site of papain by reaction of the active site thiol group with 4-chloromercuri-2-nitrophenol ( 1a), 2-chloromercuri-4-nitrophenol ( 2a), and 2-chloromercuri-4,6-dinitrophenol ( 3a) to form papain derivatives 1c–3c. Spectrophotometric titrations of these derivatives of papain yielded p K values (25 °C, Γ 2 0.15) of 7.51, 7.99, and 3.93, for the reporter groups in 1c, 2c and 3c, respectively. Comparison of these p K values with those of the corresponding 2-mercaptoethanol derivatives 1b–3b indicates that the environment at the active site of papain caused upward perturbations of 0.35 and 0.96 unit in the p K's of the reporter groups in 1c and 2c, respectively, and a downward perturbation of 0.53 unit in the p K of the reporter group in 3c. These p K perturbations were compared to those determined previously for the nitrophenol reporter groups introduced at the active site of papain by reaction of papain with α-bromo-4-hydroxy-3-nitroacetophenone and 2-bromoacetamido-4-nitrophenol. Assuming that the phenolic function of geometrically similar reporter groups occupy similar positions at the active site of the enzyme, the observed p K perturbations exhibited by these five reporter groups indicate that the charge in the vicinity of the active site of papain (a) does not change between pH 5 and 8, and (b) becomes positive at pH values below pH 4–5. These conclusions are consistent with the view that the p K values of His-159 and Asp-158 at the active site of papain are both about 4 in forms of papain wherein the active site thiolate anion is neutralized by protonation or is blocked by chemical modification.