Kinetic isotope effects and medium effects have been measured for sulfuryl-transfer reactions of the sulfate ester p-nitrophenyl sulfate (pNPS). The results are compared to those from previous studies of phosphoryl transfer, a reaction with mechanistic similarities. The N-15 and the bridge O-18 isotope effects for the reaction of the pNPS anion are very similar to those of the p-nitrophenyl phosphate (pNPP) dianion. This indicates that in the transition states for both reactions the leaving group bears nearly a full negative charge resulting from a large degree of bond cleavage to the leaving group. The nonbridge O-18 isotope effects support the notion that the sulfuryl group resembles SO(3) in the transition state. The reaction of the neutral pNPS species in acid solution is mechanistically similar to the reaction of the pNPP monoanion. In both cases proton transfer from a nonbridge oxygen atom to the leaving group is largely complete in the transition state. Despite their mechanistic similarities, the phosphoryl- and sulfuryl-transfer reactions differ markedly in their response to medium effects. Increasing proportions of the aprotic solvent DMSO to aqueous solutions of pNPP cause dramatic rate accelerations of up to 6 orders of magnitude, but only a 50-fold rate increase is observed for pNPS. Similarly, phosphoryl transfer from the pNPP dianion to tert-amyl alcohol is 9000-fold faster than the aqueous reaction, while the sulfuryl transfer from the pNPS anion is some 40-fold slower. The enthalpic and entropic contributions to these differing medium effects have been measured and compared.

General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.

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-state 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 Ala 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.