TRANSITION-STATE STRUCTURES FOR THE NATIVE DUAL-SPECIFIC PHOSPHATASE VHR AND D92N AND S131A MUTANTS, CONTRIBUTIONS TO THE DRIVING-FORCE FORCATALYSIS

Isotope effects have been measured for the reaction of the human dual- specific phosphatase VHR with p-nitrophenyl phosphate (pNPP). Isotope effects in the nonbridge oxygen atoms, in the bridge oxygen atom, and in the nitrogen atom were measured by the competitive method using an isotope ratio mass spectrometer, These are isotope effects on V/K, and give information on the chemical step of phosphoryl transfer from sub strate to the enzymatic nucleophile Cys-124, With native VHR, (18)(V/K )(nonbridge) = 1.0003 +/- 0.0003, (18)(V/K)(bridge) = 1.0118 +/- 0.002 0, and (15)(V/K) = 0.9999 +/- 0.0004. The values are similar to the in trinsic isotope effects for the uncatalyzed reaction, indicating that the chemical step is rate-limiting with the pNPP substrate, The transi tion-state structure resembles that for the uncatalyzed reaction and t hose previously found for the protein-tyrosine phosphatases YOP51 and PTP1, and is highly dissociative with P-O bond cleavage and protonatio n of the leaving group by the general acid Asp-92 both well advanced, The D92N mutant exhibits a transition state similar to that of the unc atalyzed reaction of the pNPP dianion, dissociative and with the leavi ng group departing as the nitrophenolate anion. The S131A mutation cau ses an increase in the pK(a) of the nucleophilic Cys, but the isotope effect data are unchanged from those for the native enzyme, indicating no effects of this increase in nucleophilicity on transition-state st ructure. The double mutant D92N/S131A manifests both the increase in p K(a) of the nucleophilic Cys and the loss of general acid assistance t o the leaving group. In the absence of the general acid, the change in nucleophile pK(a) results in an increase in (18)(V/K)(nonbridge) from 1.0019 (with D92N) to 1.0031 (with D92N/S131A), indicating loss of P- O nonbridge bond order in the transition state. It is concluded that t his is more likely caused by electrostatic effects rather than resulti ng from increased nucleophile-phosphorus bonding in a less dissociativ e transition state, although the latter explanation cannot be excluded on the basis of the present data. Electrostatic effects between the t hiolate anion nucleophile and the phosphoryl group may be an important part of the driving force far catalysis in this family of enzymes.