EXAMINATION OF THE TRANSITION-STATE OF THE LOW-MOLECULAR-MASS SMALL TYROSINE PHOSPHATASE .1. COMPARISONS WITH OTHER PROTEIN PHOSPHATASES

The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D12 8E, and S18A have been studied by measurement of heavy-atom isotope ef fects in the substrate. The isotope effects were measured at the nonbr idging oxygen atoms [(18)(V/K)(nonbridge)], at the bridging oxygen ato m (the site of bond cleavage) [(18)(V/K)(bridge)], and at the nitrogen atom in the nitrophenol leaving group [(15)(V/K)]. The results with n ative Stp1 were 1.0160 +/- 0.0005 for (18)(V/K)(bridge), 1.0007 +/- 0. 0001 for (15)(V/K), and 1.0018 +/- 0.0003 for (18)(V/K)(nonbridge). Th e values for (18)(V/K)(nonbridge) and (15)(V/K) differ from those prev iously measured with other protein-tyrosine phosphatases and from thos e of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interactio n between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to th e leaving group. Mutation of the general acid from aspartic acid to gl utamic acid slows catalysis but causes no change in the isotope effect s and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagin e or alanine give values for 18(V/K)(bridge) Of about 1.029, for (15)( V/K) of about 1.003, and for (18)(V/K)(nonbridge) Of 1.0010 (D128A) to 1.0024 (D128N), These data indicate a dissociative transition state w ith the leaving group departing as the nitrophenolate anion and indica te more nucleophilic participation than in the aqueous hydrolysis of t he pNPP dianion, just as in the native enzyme, The isotope effects wit h the S18A mutant, in which a hydrogen bonding stabilization of the an ionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from PNPP.