Abstract
The kinetic mechanism of the hydrolysis of phosphate monoesters catalyzed by a soluble form of rat protein-tyrosine phosphatase (PTPase), PTP1, was probed with a variety of steady-state and pre-steady-state kinetic techniques. Product inhibition and 18O exchange experiments are consistent with the enzymatic reaction proceeding through two chemical steps, i.e. formation and breakdown of a covalent phosphoenzyme intermediate. The variation of kcat/Km with pH indicates that three ionizable groups are involved in enzyme substrate binding and catalysis. The first group must be deprotonated and is attributed to the second ionization of the substrate. The other two groups with pK alpha values of 5.1 and 5.5 correspond to two enzyme active site residues. The kcat-pH profiles for both p-nitrophenyl phosphate and beta-naphthyl phosphate are bell-shaped and are superimposable, with the apparent pK alpha values derived from the acidic limb and the basic limb of the profile being 4.4 and 6.8, respectively. This suggests that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate at all pH values. Results from leaving group dependence of kcat at two different pH values support the above conclusion. Furthermore, burst kinetics have been demonstrated with PTP1 using p-nitrophenyl phosphate as a substrate. The rate constants for the formation and the breakdown of the intermediate are 241 and 12 s-1, respectively, at pH 6.0 and 3.5 degrees C. A normal D2O solvent isotope effect (kcatH/kcatD = 1.5) is associated with the breakdown of the phosphoenzyme intermediate, indicating a solvent-derived proton in the transition state. The leaving group dependence of kcat/Km suggests that there is a strong electrophilic interaction between the enzyme and the leaving group oxygen in the transition state of the phosphorylation event. These results are compared with those of the Yersinia PTPase and suggest that the mechanism for PTPase-catalyzed phosphate monoester hydrolysis is conserved from bacterial to mammals.
Highlights
Zhong-Yin Zhang:j: From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
The kinetic mechanism of the hydrolysis of phosphate monoesters catalyzed by a soluble form of rat proteintyrosine phosphatase (PTPase), PTP1, was probed with a variety of steady-state and pre-steady-state kinetic techniques
Increasing ionic strength from 0.026 to 1.35 M resulted in a 2-fold decrease in kcat value and about a 73-fold increase in Km' the effect seemed to be mainly on the apparent enzyme substrate dissociation constant Km' The Yersinia PTPase-catalyzed hydrolysis of pNPP was very sensitive to the ionic strength of the reaction medium [8]
Summary
Since the Yersinia PTPase shares only 20-30% sequence identity to mammalian PTPases in the catalytic domain [6], and its keat value is orders of magnitude higher than mammalian enzymes [8], we feel that a thorough kinetic and mechanistic study on a mammalian PTPase-catalyzed reaction is necessary. This will enable us to make a direct comparison and determine if a common catalytic mechanism is utilized by PTPases from prokaryotes to mammals.
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