Kinetic and mechanistic characterization of a mammalian protein-tyrosine phosphatase, PTP1

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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 k(cat)/K(m) 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(a) values of 5.1 and 5.5 correspond to two enzyme active site residues. The k(cat)-pH profiles for both p-nitrophenyl phosphate and β-naphthyl phosphate are bell-shaped and are superimposable, with the apparent pK(a) 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 k(cat) 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 °C. A normal D2O solvent isotope effect (k(cat)/(H)/k(cat)/(D) = 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 k(cat)/K(m) 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.

Original languageEnglish (US)
Pages (from-to)11199-11204
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number19
DOIs
StatePublished - 1995
Externally publishedYes

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Protein Tyrosine Phosphatases
Kinetics
Hydrolysis
Substrates
Enzymes
Phosphates
Phosphorylation
Mammals
Isotopes
Extremities
Catalysis
Ionization
Protons
Rats
Rate constants
Yersinia
Oxygen
Decomposition
Catalytic Domain
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and mechanistic characterization of a mammalian protein-tyrosine phosphatase, PTP1. / Zhang, Zhong-Yin.

In: Journal of Biological Chemistry, Vol. 270, No. 19, 1995, p. 11199-11204.

Research output: Contribution to journalArticle

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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 k(cat)/K(m) 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(a) values of 5.1 and 5.5 correspond to two enzyme active site residues. The k(cat)-pH profiles for both p-nitrophenyl phosphate and β-naphthyl phosphate are bell-shaped and are superimposable, with the apparent pK(a) 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 k(cat) 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 °C. A normal D2O solvent isotope effect (k(cat)/(H)/k(cat)/(D) = 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 k(cat)/K(m) 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.",
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