Substrate specificity of the protein tyrosine phosphatases

Zhong Yin Zhang, Andrea M. Thieme-Sefler, Derek Maclean, Dennis J. Mcnamara, Ellen M. Dobrusin, Tomi K. Sawyer, Jack E. Dixon

Research output: Contribution to journalArticle

174 Scopus citations

Abstract

The substrate specificity of a recombinant protein tyrosine phosphatase (PTPase) was probed using synthetic phosphotyrosine-containing peptides corresponding to several of the autophosphorylation sites in epidermal growth factor receptor (EGFR). The peptide corresponding to the autophosphorylation site, EGFR988-998, was chosen for further study due to its favorable kinetic constants. The contribution of individual amino acid side chains to the binding and catalysis was ascertained utilizing a strategy in which each amino acid within the undecapeptide EGFR988-998 (DADEpYLIPQQG) was sequentially substituted by an Ala residue (Ala-scan). The resulting effects due to singular Ala substitution were assessed by kinetic analysis with two widely divergent homogeneous PTPases. A "consensus sequence" for PTPase recognition may be suggested from the Ala-scan data as DADEpYAAPA, and the presence of acidic residues proximate to the NH2-terminal side of phosphorylation is critical for high-affinity binding and catalysis. The Km value for EGFR988-998 decreased as the pH increased, suggesting that phosphate dianion is favored for substrate binding. The results demonstrate that chemical features in the primary structure surrounding the dephosphorylation site contribute to PTPase substrate specificity.

Original languageEnglish (US)
Pages (from-to)4446-4450
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number10
DOIs
StatePublished - May 15 1993
Externally publishedYes

Keywords

  • Enzyme kinetics

ASJC Scopus subject areas

  • General

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    Zhang, Z. Y., Thieme-Sefler, A. M., Maclean, D., Mcnamara, D. J., Dobrusin, E. M., Sawyer, T. K., & Dixon, J. E. (1993). Substrate specificity of the protein tyrosine phosphatases. Proceedings of the National Academy of Sciences of the United States of America, 90(10), 4446-4450. https://doi.org/10.1073/pnas.90.10.4446