title: The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase.
abstract: 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 Asp(50) to alanine or Arg(49) 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 Arg(49) and Asp(50) may be targeted for the design of potent and selective inhibitors of TCPTP and PTP1B.
authors.affiliation: Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, Pointe-Claire-Dorval H9R 4P8, Canada. Ernest_Asanteappiah@merck.com
authors.forename: E
authors.initials: E
authors.lastname: Asante-Appiah
authors.forename: K
authors.initials: K
authors.lastname: Ball
authors.forename: K
authors.initials: K
authors.lastname: Bateman
authors.forename: K
authors.initials: K
authors.lastname: Skorey
authors.forename: R
authors.initials: R
authors.lastname: Friesen
authors.forename: C
authors.initials: C
authors.lastname: Desponts
authors.forename: P
authors.initials: P
authors.lastname: Payette
authors.forename: C
authors.initials: C
authors.lastname: Bayly
authors.forename: R
authors.initials: R
authors.lastname: Zamboni
authors.forename: G
authors.initials: G
authors.lastname: Scapin
authors.forename: C
authors.initials: C
authors.lastname: Ramachandran
authors.forename: B P
authors.initials: BP
authors.lastname: Kennedy
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D006801:Humans; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology*
chemical_list: D017027:Protein Tyrosine Phosphatases