Abstract

Purple acid phosphatases (PAPs) are dinuclear monoesterases1,2 that are structurally diverse but with a conserved set of ligands to the Fe3+-M2+ metal centers (M2+ ) Fe, Zn, or Mn).3 The identity of the nucleophile and the substrate binding mode have been matters of controversy (Figure 1).1,3-5 In one proposal, substrate binds to the divalent metal and is attacked by a terminal Fe3+-bound hydroxide. In an alternative mechanism, substrate coordinates first to the divalent metal and then forms a bridging complex, followed by nucleophilic attack by the í-hydroxide. We report that the PAPs from pig and kidney bean catalyze the hydrolysis of diesters if the second ester group is small, and the kinetics of the reaction with methyl p-nitrophenylphosphate suggest that a terminal-bound hydroxide is the nucleophile for the diester, followed by attack of the bridging hydroxide upon the resulting monoester without release into solution.