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

172 Citations (Scopus)

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
StatePublished - May 15 1993
Externally publishedYes

Fingerprint

Protein Tyrosine Phosphatases
Substrate Specificity
Catalysis
Amino Acids
Peptides
Phosphotyrosine
Consensus Sequence
Recombinant Proteins
Epidermal Growth Factor Receptor
Phosphates
Phosphorylation

Keywords

  • Enzyme kinetics

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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.

Substrate specificity of the protein tyrosine phosphatases. / Zhang, Zhong-Yin; Thieme-Sefler, Andrea M.; Maclean, Derek; Mcnamara, Dennis J.; Dobrusin, Ellen M.; Sawyer, Tomi K.; Dixon, Jack E.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, No. 10, 15.05.1993, p. 4446-4450.

Research output: Contribution to journalArticle

Zhang, Z-Y, Thieme-Sefler, AM, Maclean, D, Mcnamara, DJ, Dobrusin, EM, Sawyer, TK & Dixon, JE 1993, 'Substrate specificity of the protein tyrosine phosphatases', Proceedings of the National Academy of Sciences of the United States of America, vol. 90, no. 10, pp. 4446-4450.
Zhang Z-Y, Thieme-Sefler AM, Maclean D, Mcnamara DJ, Dobrusin EM, Sawyer TK et al. Substrate specificity of the protein tyrosine phosphatases. Proceedings of the National Academy of Sciences of the United States of America. 1993 May 15;90(10):4446-4450.
Zhang, Zhong-Yin ; Thieme-Sefler, Andrea M. ; Maclean, Derek ; Mcnamara, Dennis J. ; Dobrusin, Ellen M. ; Sawyer, Tomi K. ; Dixon, Jack E. / Substrate specificity of the protein tyrosine phosphatases. In: Proceedings of the National Academy of Sciences of the United States of America. 1993 ; Vol. 90, No. 10. pp. 4446-4450.
@article{7b31134a92d84efc9a15bf12049d17d5,
title = "Substrate specificity of the protein tyrosine phosphatases",
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.",
keywords = "Enzyme kinetics",
author = "Zhong-Yin Zhang and Thieme-Sefler, {Andrea M.} and Derek Maclean and Mcnamara, {Dennis J.} and Dobrusin, {Ellen M.} and Sawyer, {Tomi K.} and Dixon, {Jack E.}",
year = "1993",
month = "5",
day = "15",
language = "English (US)",
volume = "90",
pages = "4446--4450",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "10",

}

TY - JOUR

T1 - Substrate specificity of the protein tyrosine phosphatases

AU - Zhang, Zhong-Yin

AU - Thieme-Sefler, Andrea M.

AU - Maclean, Derek

AU - Mcnamara, Dennis J.

AU - Dobrusin, Ellen M.

AU - Sawyer, Tomi K.

AU - Dixon, Jack E.

PY - 1993/5/15

Y1 - 1993/5/15

N2 - 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.

AB - 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.

KW - Enzyme kinetics

UR - http://www.scopus.com/inward/record.url?scp=0027174567&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027174567&partnerID=8YFLogxK

M3 - Article

C2 - 7685104

AN - SCOPUS:0027174567

VL - 90

SP - 4446

EP - 4450

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 10

ER -