Catalytic function of the conserved hydroxyl group in the protein tyrosine phosphatase signature motif.
Burst kinetics is observed with the Yersinia protein tyrosine phosphatase (PTPase). This provides direct kinetic evidence for a phosphoenzyme mechanism and suggests that the breakdown of the phosphoenzyme intermediate is the rate-limiting step. Burst kinetics is a powerful tool for mechanistic studies of PTPase catalysis since functional roles of active site residues can be evaluated by studying their effects on the individual elementary steps associated with the formation and the breakdown of the intermediate. In order to investigate the role of Thr410, a conserved residue that is present in the PTPase signature motif, this residue was altered by site-directed mutagenesis to serine and alanine. 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 plays an important role in facilitating the breakdown of the phosphoenzyme intermediate.
Zhang
Z Y
ZY
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Palfey
B A
BA
Wu
L
L
Zhao
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D015007:Yersinia / Q000201:enzymology*
D007461:Iodoacetates; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D013912:Threonine; C008644:nitrophenylphosphate; D017027:Protein Tyrosine Phosphatases; D019807:Iodoacetic Acid