| Abstract
| - The reactions of p-nitrophenyl phosphate(pNPP) with the low-molecular mass tyrosinephosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18Ahave been studied bymeasurement of heavy-atom isotope effects in the substrate. Theisotope effects were measured at thenonbridging oxygen atoms[18(V/K)nonbridge],at the bridging oxygen atom (the site of bondcleavage)[18(V/K)bridge],and at the nitrogen atom in the nitrophenol leaving group[15(V/K)]. The resultswith nativeStp1 were 1.0160 ± 0.0005 for18(V/K)bridge,1.0007 ± 0.0001 for 15(V/K),and 1.0018 ± 0.0003 for18(V/K)nonbridge.The values for18(V/K)nonbridge and15(V/K) differ from thosepreviously measured withother protein-tyrosine phosphatases and from those of the aqueoushydrolysis reaction of pNPP. Thevalues indicate that in the transition state of the native Stp1reaction the leaving group bears a partialnegative charge, and there is nucleophilic interaction between the Cysnucleophile, and the phosphorylgroup, causing some decrease in the nonbridge P−O bond order.The transition state remains highlydissociative with respect to the degree of bond cleavage to the leavinggroup. Mutation of the generalacid from aspartic acid to glutamic acid slows catalysis but causes nochange in the isotope effects andthus does not alter the degree of proton transfer to the leaving groupin the transition state. Mutations ofthis residue to asparagine or alanine give values for18(V/K)bridge ofabout 1.029, for 15(V/K) ofabout1.003, and for18(V/K)nonbridge of1.0010 (D128A) to 1.0024 (D128N). These data indicate adissociativetransition state with the leaving group departing as the nitrophenolateanion and indicate more nucleophilicparticipation than in the aqueous hydrolysis of the pNPPdianion, just as in the native enzyme. Theisotope effects with the S18A mutant, in which a hydrogen bondingstabilization of the anionic Cysnucleophile has been removed, were within experimental error of thosewith the native enzyme, indicatingthat this alteration has no effect on the transition state forphosphoryl transfer from pNPP.
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