The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase

We have studied T-cell protein-tyrosine phosphatase (TCPTP) as a model phos phatase in an attempt to unravel amino acid residues that may influence the design of specific inhibitors. Residues 48-50, termed the YRD motif, a reg ion that is found in protein-tyrosine phosphatases, but absent in dual-spec ificity phosphatases was targeted. YRD derivatives of TCPTP were characteri zed by steady-state kinetics and by inhibition studies with BzN-EJJ-amide, a potent inhibitor of TCPTP, Substitution of Asp(50) to alanine or Arg(49) to lysine, methionine, or alanine significantly affected substrate hydrolys is and led to a substantial decrease in affinity for BzN-EJJ-amide, The inf luence of residue 49 on substrate/inhibitor selectivity was further investi gated by comparing subsite amino acid preferences of TCPTP and its R49K der ivative by affinity selection coupled with mass spectrometry, The greatest effect on selectivity was observed on the residue that precedes the phospho rylated tyrosine, Unlike wild-type TCPTP, the R49K derivative preferred tyr osine to aspartic or glutamic acid. BzN-EJJ-amide which retains the preferr ed specificity requirements of TCPTP and PTP1B was equipotent on both enzym es but greater than 30-fold selective over other phosphatases, These result s suggest that Ar-49 and Asp(50) may be targeted for the design of potent a nd selective inhibitors of TCPTP and PTP1B.