Substrate specificity of the protein tyrosine phosphatases.
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.
Zhang
Z Y
ZY
Department of Biological Chemistry, Medical School, Walther Cancer Institute, University of Michigan, Ann Arbor 48109.
Thieme-Sefler
A M
AM
Maclean
D
D
McNamara
D J
DJ
Dobrusin
E M
EM
Sawyer
T K
TK
Dixon
J E
JE
D000595:Amino Acid Sequence; D066246:ErbB Receptors / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D015007:Yersinia / Q000201:enzymology
D009842:Oligopeptides; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D017027:Protein Tyrosine Phosphatases