MAPPING THE SUBSITE PREFERENCES OF PROTEIN-TYROSINE-PHOSPHATASE PTP-1B USING COMBINATORIAL CHEMISTRY APPROACHES

Protein tyrosine phosphatases (PTPases) are important regulators of si gnal transduction systems, but the specificity of their action is larg ely unexplored. We have approached this problem by attempting to map t he subsite preferences of these enzymes using combinatorial chemistry approaches. Protein-tyrosine peptidomimetics containing nonhydrolyzabl e phosphotyrosine analogues bind to PTPases with high affinity and act as competitive inhibitors of phosphatase activity. Human PTP-1B, a PT Pase implicated to play an important role in the regulation of growth factor signal transduction pathways, was used to screen a synthetic co mbinatorial library containing malonyltyrosine as a phosphotyrosine mi mic. Using two cross-validating combinatorial chemistry screening appr oaches, one using an iterative method and the ether employing library affinity selection-mass spectrometric detection, peptides with high af finity for PTP-1B were identified and subsite preferences were detaile d by quantitatively comparing residues of different character. Consist ent with previous observations, acidic residues were preferred in subs ites X-3 and X-2 In contrast, aromatic substitutions were clearly pref erred at the X-1 subsite. This information supports the concept that t his class of enzymes may have high substrate specificity as dictated b y the sequence proximal to the phosphorylation site. The results are d iscussed with regards to the use of combinatorial techniques in order to elucidate the interplay between enzyme subsites.