DETERMINANTS OF SUBSTRATE RECOGNITION IN THE PROTEIN-TYROSINE-PHOSPHATASE, PTP1

Photoaffinity labeling has been used to identify amino acids involved in recognition of protein substrates by the protein-tyrosine phosphata se PTP1, The photoactive amino acid p-benzoylphenylalanine (Bpa) was i ncorporated into a phosphotyrosine containing peptide derived from epi dermal growth factor autophosphorylation site Tyr(992) (EGFR(988-998)) . This peptide photoinactivated PTP1 in a time- and concentration-depe ndent manner. Three lines of evidence indicate that the interaction be tween PTP1 and the photoaffinity label was specific: 1) photoinactivat ion was inhibited in the presence of a non-Bpa-containing peptide from EGFR Tyr(992) in molar excess, 2) The photoaffinity label-containing phosphopeptide was rapidly dephosphorylated by PTP1 with kinetic const ants similar to those of the non-Bpa-containing peptide under identica l conditions. 3) After complete photoinactivation, the level of incorp oration of radioactive photoaffinity label into PTP1 was approximately 0.9 mol of label/mol of enzyme, consistent with a 1:1 stoichiometry o f photolabeling. Radiolabeled peptide was used to identify sites of cr oss-linking to PTP1. Bpa peptide-PTP1 was digested with trypsin, and r adioactive fragments were purified by high performance liquid chromato graphy (HPLC) and analyzed by Edman sequencing. In two parallel experi ments which were analyzed using different HPLC columns, a site in the alpha 2' region of PTP1, most likely Ile(23), was labeled by the Tyr(9 92)-derived peptide. The results are discussed in light of the crystal structure of human PTP1B and suggest that an additional mode of subst rate recognition must exist for PTP1 catalysis.