Transition-state structures for the native dual-specific phosphatase VHR and D92N and S131A mutants. Contributions to the driving force for catalysis.

Abstract

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 substrate to the enzymatic nucleophile Cys-124. With native VHR, 18(V/K)nonbridge = 1.0003 +/- 0.0003, 18(V/K)bridge = 1.0118 +/- 0.0020, and 15(V/K) = 0.9999 +/- 0.0004. The values are similar to the intrinsic isotope effects for the uncatalyzed reaction, indicating that the chemical step is rate-limiting with the pNPP substrate. The transition-state structure resembles that for the uncatalyzed reaction and those previously found for the protein-tyrosine phosphatases YOP51 and PTP1, and is highly dissociative with P-O bond cleavage and protonation 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 uncatalyzed reaction of the pNPP dianion, dissociative and with the leaving group departing as the nitrophenolate anion. The S131A mutation causes an increase in the pKa 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 structure. The double mutant D92N/S131A manifests both the increase in pKa of the nucleophilic Cys and the loss of general acid assistance to the leaving group. In the absence of the general acid, the change in nucleophile pKa 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 this is more likely caused by electrostatic effects rather than resulting from increased nucleophile-phosphorus bonding in a less dissociative transition state, although the latter explanation cannot be excluded on the basis of the present data. Electrostatic effects between the thiolate anion nucleophile and the phosphoryl group may be an important part of the driving force for catalysis in this family of enzymes.