Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors.
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.
Cho
H
H
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Krishnaraj
R
R
Itoh
M
M
Kitas
E
E
Bannwarth
W
W
Saito
H
H
Walsh
C T
CT
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D009842:Oligopeptides; D010446:Peptide Fragments; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases