Positive charge is uniformly present in the active sites of all known phosphatases. The postulate that this charge imparts a change to the mechanism and the transition state for phosphoryl transfer was examined by comparing kinetic isotope effects with the substrate p-nitrophenyl phosphate for reactions of the native protein tyrosine phosphatase from Yersinia with data from mutants in which the conserved arginine residue was mutated to Lys or to Ala. The k(cat),,, values for both mutants are about 10(4) less than that of the native enzyme but are still nearly 10(5)-fold faster than the uncatalyzed rate. Steady-slate kinetic data as well as isotope effects showed that both mutations interfere with functioning of general acid catalysis. To examine the effect of positive charge on the transition state free of this additional effect, double mutants were made in which general acid catalysis was removed by mutation of Asp356 to either Asn or Al in addition to the mutation to Arg. The k(cat)/K-m values of D356A and D356N are 300-360-fold higher than those of R409A/D356A and R409A/D356N suggesting that the side chain of Arg409 contributes 3.4-3.5 kcal/mol to transition-state stabilization. Comparisons of the isotope effects for reactions of the double mutants with data from general acid single mutants show that mutation of Arg to either Lys or to Ala does not significantly affect the transition state for phosphoryl transfer. This indicates that this residue functions to stabilize the transition state but does not alter it from its structure in the uncatalyzed reaction.