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J. Biol. Chem., Vol. 276, Issue 28, 26036-26043, July 13, 2001

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The YRD Motif Is a Major Determinant of Substrate and Inhibitor Specificity in T-cell Protein-tyrosine Phosphatase^*

Ernest Asante-Appiah^§, Kristen Ball, Kevin Bateman^¶, Kathryn Skorey, Rick Friesen^¶, Caroline Desponts, Paul Payette, Christopher Bayly^¶, Robert Zamboni^¶, Giovanna Scapin, Chidambaram Ramachandran, and Brian P. Kennedy

From the  Department of Biochemistry and Molecular Biology and the ^¶ Department of Medicinal Chemistry, Merck Frosst Center for Therapeutic Research, Pointe-Claire-Dorval H9R 4P8, Canada and the  Department of Endocrinology and Cell Biology, Merck Research Laboratory, Rahway, New Jersey 07065

We have studied T-cell protein-tyrosine phosphatase (TCPTP) as a model phosphatase in an attempt to unravel amino acid residues that may influence the design of specific inhibitors. Residues 48-50, termed the YRD motif, a region that is found in protein-tyrosine phosphatases, but absent in dual-specificity phosphatases was targeted. YRD derivatives of TCPTP were characterized by steady-state kinetics and by inhibition studies with BzN-EJJ-amide, a potent inhibitor of TCPTP. Substitution of Asp⁵⁰ to alanine or Arg⁴⁹ to lysine, methionine, or alanine significantly affected substrate hydrolysis and led to a substantial decrease in affinity for BzN-EJJ-amide. The influence of residue 49 on substrate/inhibitor selectivity was further investigated by comparing subsite amino acid preferences of TCPTP and its R49K derivative by affinity selection coupled with mass spectrometry. The greatest effect on selectivity was observed on the residue that precedes the phosphorylated tyrosine. Unlike wild-type TCPTP, the R49K derivative preferred tyrosine to aspartic or glutamic acid. BzN-EJJ-amide which retains the preferred specificity requirements of TCPTP and PTP1B was equipotent on both enzymes but greater than 30-fold selective over other phosphatases. These results suggest that Arg⁴⁹ and Asp⁵⁰ may be targeted for the design of potent and selective inhibitors of TCPTP and PTP1B.

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