Leaving group dependence and proton inventory studies of the phosphorylation of a cytoplasmic phosphotyrosyl protein phosphatase from bovine heart

Zhong-Yin Zhang, Robert L. Van Etten

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Abstract

The kcat and Km values for the bovine heart low molecular weight phosphotyrosyl protein phosphatase catalyzed hydrolysis of 16 aryl phosphate monoesters and of five alkyl phosphate monoesters having the structure Ar(CH2)nOPO3H2 (n = 1-5) were measured at pH 5.0 and 37°C. With the exception of α-naphthyl phosphate and 2-chlorophenyl phosphate, which are subject to steric effects, the values of kcat are effectively constant for the aryl phosphate monoesters. This is consistent with the catalysis being nucleophilic in nature, with the existence of a common covalent phosphoenzyme intermediate, and with the breakdown of this intermediate being rate-limiting. In contrast, kcat for the alkyl phosphate monoesters is much smaller and the rate-limiting step for these substrates is interpreted to be the phosphorylation of the enzyme. A single linear correlation is observed for a plot of log (kcat/Km vs leaving group pKa for both classes of substrates at pH 5.0: log (kcat/Km = -0.28pKa + 6.88 (n = 19, r = 0.89), indicating a uniform catalytic mechanism for the phosphorylation event. The small change in effective charge (-0.28) on the departing oxygen of the substrate is similar to that observed in the specific acid catalyzed hydrolysis of monophosphate monoanions (-0.27) and is consistent with a strong electrophilic interaction of the enzyme with this oxygen atom in the transition state. The D2O solvent isotope effect and proton inventory experiments indicate that only one proton is "in flight" in the transition state of the phosphorylation process and that this proton transfer is responsible for the reduction of effective charge on the leaving oxygen.

Original languageEnglish (US)
Pages (from-to)8954-8959
Number of pages6
JournalBiochemistry
Volume30
Issue number37
StatePublished - 1991
Externally publishedYes

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Phosphorylation
Protein Tyrosine Phosphatases
Protons
Phosphates
Equipment and Supplies
Oxygen
Hydrolysis
Substrates
Proton transfer
Enzymes
Catalysis
Isotopes
Molecular Weight
Molecular weight
Atoms
Acids
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Leaving group dependence and proton inventory studies of the phosphorylation of a cytoplasmic phosphotyrosyl protein phosphatase from bovine heart. / Zhang, Zhong-Yin; Van Etten, Robert L.

In: Biochemistry, Vol. 30, No. 37, 1991, p. 8954-8959.

Research output: Contribution to journalArticle

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