MOLECULAR-BASIS FOR SUBSTRATE-SPECIFICITY OF PROTEIN-TYROSINE-PHOSPHATASE 1B

Protein-tyrosine phosphatases can exhibit stringent substrate specific ity in vivo, although the molecular basis for this is not well underst ood. The three-dimensional structure of the catalytically inactive pro tein-tyrosine phosphate 1B (PTP1B)/C215S complexed with an optimal sub strate, DADEpYL-NH2, reveals specific interactions between amino acid residues in the substrate and PTP1B. The goal of this work is to rigor ously evaluate the functional significance of Tyr(46), Arg(47), Asp(48 ) Phe(182), and Gln(262) in substrate binding and catalysis, using sit e-directed mutagenesis. Combined with structural information, kinetic analysis of the wild type and mutant PTP1B using p-nitrophenyl phospha te and phosphotyrosine-containing peptides has yielded further insight into PTP1B residues, which recognize general features, as well, as sp ecific properties, in peptide substrates, In addition, the kinetic res ults suggest roles of these residues in E-P hydrolysis, which are not obvious from the structure of PTP1B/peptide complex. Thus, Tyr(46) and Asp(48) recognize common features of peptide substrates and are impor tant for peptide substrate binding and/or E-P formation. Arg47 acts as a determinant of substrate specificity and is responsible for the mod est preference of PTP1B for acidic residues NH2-terminal to phosphotyr osine, Phe(182) and the invariant Gln2G2 are not only important for su bstrate binding and/or E-P formation but also important for the E-P hy drolysis step.