title: The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding.
abstract: Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases.
authors.affiliation: Metabolic and Cardiovascular Diseases Research, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Picha
authors.forename: Smita S
authors.initials: SS
authors.lastname: Patel
authors.forename: Sreekala
authors.initials: S
authors.lastname: Mandiyan
authors.forename: James
authors.initials: J
authors.lastname: Koehn
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: Wennogle
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D017931:DNA Primers; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases