Insights from heavy-atom isotope effects on phosphoryl and thiophosphoryl transfer reactions

Phosphatases are extremely efficient enzymes, with k(cat) values many orders of magnitude faster than the rate constants for the uncatalyzed hydrolysis of their normally unreactive phosphate monoester substrates. There is considerable interest in whether these enzymatic mechanisms and transition states for monoester hydrolysis differ from the uncatalyzed reactions in solution. We have used heavy atom isotope effects to examine and compare a number of enzymatic and uncatalyzed phosphoryl transfer reactions of p-nitrophenyl phosphate and p-nitrophenyl phosphorothioate. Isotope effects for several protein tyrosine phosphatases and dual-specific phosphatases reveal that they utilize dissociative transition stares similar to the uncatalyzed reaction, with proton transfer to the leaving group either complete or nearly so in the transition state for each of the enzymes studied. The general acid mutants of these enzymes are still competent catalysts but exhibit a hybrid transition state, having slightly increased nucleophilic participation but with the same extensive bond cleavage to the leaving group. Ser/Thr phosphatases have binuclear metal centers and transfer the phosphoryl group to a metal-bound water molecule. The bacteriophage Lambda phosphatase, a member of this family, exhibits isotope effects also consistent with a dissociative transition state. His-76 has been proposed to serve as a general acid, and the isotope effect data with the mutant H76N show that the leaving group bears substantially more negative charge in the transition state.