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 synthet ic phosphotyrosine (pY)-containing peptides corresponding to the autop hosphorylation site in EGF receptor (EGFR) at Y992. The peptide EGFR(9 88-998) (DADEpYLIPQQG) is chosen as a template due to its favorable ki netic constants. The contribution of individual amino acids on both si des of pY to binding and catalysis was assessed by kinetic analysis us ing a continuous, spectrophotometric assay. For both Yersinia PTPase a nd a soluble recombinant mammalian PTPase of 323 amino acid residues ( rat PTP1), efficient binding and catalysis required six amino acids in cluding the pY residue, i.e., four residues N-terminal to pY and one r esidue C-terminal to pY. Thus, PTPase substrate specificity is primari ly dictated by residues to the N-terminal side of pY. The pY moiety an d the rest of the peptide interact with PTPases in a cooperative manne r. The presence of pY in the peptide substrate is necessary but not su fficient for high-affinity binding, since phosphotyrosine and other si mple aryl phosphates exhibit weak binding, and dephosphorylated peptid es do not bind to PTPases. Two variations on the pY moiety are also ex amined in order to assess their utility in PTPase inhibitor design. It is demonstrated that the thiophosphoryl analog in which one of the ph osphate oxygens is replaced by sulfur can be hydrolyzed by PTPases, wh ereas the phosphonomethylphenylalanine analog in which the tyrosyl oxy gen is replaced by a CH2 group is a competitive and nonhydrolyzable in hibitor, with K-i values of 18.6 and 10.2 mu M, respectively, for the Yersinia PTPase and the rat PTP1.