Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors

Protein Sci. 1993 Jun;2(6):977-84. doi: 10.1002/pro.5560020611.

Abstract

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.

MeSH terms

  • Amino Acid Sequence
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Humans
  • Kinetics
  • Leukocyte Common Antigens / genetics
  • Leukocyte Common Antigens / metabolism
  • Molecular Sequence Data
  • Oligopeptides / chemistry
  • Oligopeptides / metabolism
  • Oligopeptides / pharmacology
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Phosphorylation
  • Protein Tyrosine Phosphatases / antagonists & inhibitors
  • Protein Tyrosine Phosphatases / genetics
  • Protein Tyrosine Phosphatases / metabolism*
  • Substrate Specificity

Substances

  • Oligopeptides
  • Peptide Fragments
  • Leukocyte Common Antigens
  • Protein Tyrosine Phosphatases