Protein tyrosine phosphatase substrate specificity: size and phosphotyrosine positioning requirements in peptide substrates

摘要: 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. peptide EGFR988-998 (DADEpYLIPQQG) is chosen as a template due its favorable kinetic constants. contribution individual amino acids on both sides pY binding and catalysis was assessed by analysis continuous, spectrophotometric assay. For Yersinia PTPase soluble mammalian 323 acid residues (rat PTP1), efficient required six including residue, i.e., four N-terminal one residue C-terminal pY. Thus, substrate specificity primarily dictated side moiety rest interact with PTPases cooperative manner. presence necessary but not sufficient high-affinity binding, since other simple aryl phosphates exhibit weak dephosphorylated do bind PTPases. Two variations also examined order assess their utility inhibitor design. It demonstrated that thiophosphoryl analog which phosphate oxygens replaced sulfur can be hydrolyzed PTPases, whereas phosphonomethylphenylalanine tyrosyl oxygen CH2 group competitive nonhydrolyzable inhibitor, Ki values 18.6 10.2 microM, respectively, rat PTP1.