Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein phosphatase

Zhong-Yin Zhang, J. P. Davis, R. L. Van Etten

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Covalent modification experiments were conducted in order to identify active site residues of the 18-kDa cytoplasmic phosphotyrosyl protein phosphatases. The enzyme was inactivated by diethyl pyrocarbonate, phenylglyoxal, cyclohexanedione, iodoacetate, iodoacetamide, phenylarsine oxide, and certain epoxides in a manner consistent with the modification of active site residues. Phenylglyoxal and cyclohexanedione both bind to the active site in a rapid preequilibrium process and thus act as active site- directed inhibitors. The pH dependencies of the inactivation by iodoacetate and by iodoacetamide were examined in detail and compared with rate data for the alkylation of glutathione as a model compound. The enzyme inactivation data permitted the determination of pK(a) values of two reactive cysteines at or near the active site. Although phosphomycin is simply a competitive inhibitor of the enzyme, it was found that 1,2-epoxy-3-(p- nitrophenoxy)propane (EPNP) and (R)- and (S)-benzylglycidol act as irreversible covalent inactivators, consistent with the importance of a hydrophobic moiety on the substrate in controlling substrate specificity. EPNP exhibits characteristics of an active site-directed inactivator, with a preequilibrium binding constant somewhat smaller than that of phosphate ion. The pH dependencies of inactivation of EPNP and (S)-benzylglycidol are identical to that observed for iodoacetamide and similar to that for iodoacetate, suggesting that they modify similar groups. Sequencing of the tryptic digests of the EPNP-labeled enzyme indicates that Cys-62 and Cys-145 are labeled. Phenylarsine oxide acts as a very slow, tight-binding inhibitor of the enzyme. The results are interpreted in terms of an active site model that incorporates a histidine-cysteine ion pair, similar to that present in papain.

Original languageEnglish (US)
Pages (from-to)1701-1711
Number of pages11
JournalBiochemistry
Volume31
Issue number6
StatePublished - 1992
Externally publishedYes

Fingerprint

Iodoacetates
Iodoacetamide
Propane
Protein Tyrosine Phosphatases
Phenylglyoxal
Catalytic Domain
Molecular Weight
Molecular weight
Enzyme Inhibitors
Cysteine
Enzymes
Diethyl Pyrocarbonate
Ions
Fosfomycin
Papain
Epoxy Compounds
Alkylation
Substrates
Histidine
Glutathione

ASJC Scopus subject areas

  • Biochemistry

Cite this

Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein phosphatase. / Zhang, Zhong-Yin; Davis, J. P.; Van Etten, R. L.

In: Biochemistry, Vol. 31, No. 6, 1992, p. 1701-1711.

Research output: Contribution to journalArticle

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