CATALYTIC FUNCTION OF THE CONSERVED HYDROXYL GROUP IN THE PROTEIN-TYROSINE-PHOSPHATASE SIGNATURE MOTIF

Burst kinetics is observed with the Yersinia protein tyrosine phosphat ase (PTPase). This provides direct kinetic evidence for a phosphoenzym e mechanism and suggests that the breakdown of the phosphoenzyme inter mediate is the rate-limiting step. Burst kinetics is a powerful tool f or mechanistic studies of PTPase catalysis since functional roles of a ctive site residues can be evaluated by studying their effects on the individual elementary steps associated with the formation and the brea kdown of the intermediate. In order to investigate the role of Thr410, a conserved residue that is present in the PTPase signature motif, th is residue was altered by site-directed mutagenesis to serine and alan ine. The effects of these mutations, as observed in both steady-state and pre-steady-state kinetic experiments with p-nitrophenyl phosphate (pNPP) as a substrate, demonstrated that the hydroxyl group of Thr410 is directly involved in catalysis. The hydroxyl group at residue 410 p lays an important role in facilitating the breakdown of the phosphoenz yme intermediate.