Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia

General acid catalysis in protein tyrosine phosphatases (PTPases) is accomp lished by a conserved Asp residue, which is brought into position for catal ysis 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 t o this conformation change. Residue Trp354 is at a hinge of the loop: and A rg409 forms hydrogen bonding and ionic interactions with the phosphoryl gro up 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 s teady state kinetics and heavy-atom isotope effects with the substrate p-ni trophenyl phosphate, The data indicate that mutation of the hinge residue T rp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only part ially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete, Mutation of Arp409 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 R4 09A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but par tial proton transfer during catalysis still occurs in the Ala mutant. Struc tural explanations for the differential effects of these mutations on movem ent of the flexible loop that enables general acid catalysis are presented.