Reactivity of alcohols toward the phosphoenzyme intermediate in the protein-tyrosine phosphatase-catalyzed reaction: Probing the transition state of the dephosphorylation step

In solution phosphate monoesters hydrolyze via a highly dissociative mechanism involving a ''loose'' or ''exploded'' metaphosphate-like transition state where bond formation to the incoming nucleophile is minimal and bond breaking between phosphorus and the leaving group is substantial. To better understand how protein-tyrosine phosphatase (PTPase) effects catalysis, it is important to determine the nature of the enzymic transition state. PTPases catalyze the hydrolysis of phosphate monoesters by a two-step mechanism that proceeds through a phosphoenzyme intermediate (E-P). Extensive heavy atom kinetic isotope effect and leaving group dependency studies have provided insights into the nature of the transition state for the first step (E-P formation) of the PTPase reaction. In this paper we have probed the transition state for the low M(r)PTPase-catalyzed dephosphorylation step by studying the effect of changing the alcohol basicity on its reactivity toward E-P. The Bronsted beta(nu:) value for the reactions of alcohols and E-P is determined to be 0.14, which indicates that the enzymic transition state is highly dissociative and similar to that in uncatalyzed solution reactions. We show that the conserved hydroxyl group in the PTPase signature motif is primarily involved in the E-P dephosphorylation step. We further demonstrate that elimination of the hydroxyl group renders the transition state for E-P dephosphorylation less dissociative, suggesting that the main function of the hydroxyl group in the PTPase active site is to promote the E-P going through a dissociative pathway and to stabilize the dissociative transition state.