Protein tyrosine phosphatase substrate specificity: size and phosphotyrosine positioning requirements in peptide substrates.
The structural requirements of substrates for two recombinant protein tyrosine phosphatases (PTPases) are probed using various-sized synthetic phosphotyrosine (pY)-containing peptides corresponding to the autophosphorylation site in EGF receptor (EGFR) at Y992. The peptide EGFR988-998 (DADEpYLIPQQG) is chosen as a template due to its favorable kinetic constants. The contribution of individual amino acids on both sides of pY to binding and catalysis was assessed by kinetic analysis using a continuous, spectrophotometric assay. For both Yersinia PTPase and a soluble recombinant mammalian PTPase of 323 amino acid residues (rat PTP1), efficient binding and catalysis required six amino acids including the pY residue, i.e., four residues N-terminal to pY and one residue C-terminal to pY. Thus, PTPase substrate specificity is primarily dictated by residues to the N-terminal side of pY. The pY moiety and the rest of the peptide interact with PTPases in a cooperative manner. The presence of pY in the peptide substrate is necessary but not sufficient for high-affinity binding, since phosphotyrosine and other simple aryl phosphates exhibit weak binding, and dephosphorylated peptides do not bind to PTPases. Two variations on the pY moiety are also examined in order to assess their utility in PTPase inhibitor design. It is demonstrated that the thiophosphoryl analog in which one of the phosphate oxygens is replaced by sulfur can be hydrolyzed by PTPases, whereas the phosphonomethylphenylalanine analog in which the tyrosyl oxygen is replaced by a CH2 group is a competitive and nonhydrolyzable inhibitor, with Ki values of 18.6 and 10.2 microM, respectively, for the Yersinia PTPase and the rat PTP1.
Zhang
Z Y
ZY
Department of Biological Chemistry, Medical School, Walther Cancer Institute, University of Michigan, Ann Arbor 48109.
Maclean
D
D
McNamara
D J
DJ
Sawyer
T K
TK
Dixon
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D015007:Yersinia / Q000201:enzymology
D010446:Peptide Fragments; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D017027:Protein Tyrosine Phosphatases