1pa1

File:1pa1.jpg

spinqualitylabelsall models 1pa1, resolution 1.6Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D

Crystal structure of the C215D mutant of protein tyrosine phosphatase 1B

Overview

We have characterized the C215D active-site mutant of protein-tyrosine, phosphatase-1B (PTP-1B) and solved the crystal structure of the catalytic, domain of the apoenzyme to a resolution of 1.6 A. The mutant enzyme, displayed maximal catalytic activity at pH approximately 4.5, which is, significantly lower than the pH optimum of 6 for wild-type PTP-1B., Although both forms of the enzyme exhibited identical Km values for, hydrolysis of p-nitrophenyl phosphate at pH 4.5 and 6, the kcat values of, C215D were approximately 70- and approximately 7000-fold lower than those, of wild-type PTP-1B, respectively. Arrhenius plots revealed that the, mutant and wild-type enzymes displayed activation energies of 61 +/- 1 and, 18 +/- 2 kJ/mol, respectively, at their pH optima. Unlike wild-type, PTP-1B, C215D-mediated p-nitrophenyl phosphate hydrolysis was inactivated, by 1,2-epoxy-3-(p-nitrophenoxy)propane, suggesting a direct involvement of, Asp215 in catalysis. Increasing solvent microviscosity with sucrose (up to, 40% (w/v)) caused a significant decrease in kcat/Km of the wild-type, enzyme, but did not alter the catalytic efficiency of the mutant protein., Structurally, the apoenzyme was identical to wild-type PTP-1B, aside from, the flexible WPD loop region, which was in both "open" and "closed", conformations. At physiological pH, the C215D mutant of PTP-1B should be, an effective substrate-trapping mutant that can be used to identify, cellular substrates of PTP-1B. In addition, because of its insensitivity, to oxidation, this mutant may be used for screening fermentation broth and, other natural products to identify inhibitors of PTP-1B.

Disease

Known diseases associated with this structure: Abdominal body fat distribution, modifier of OMIM:[176885], Insulin resistance, susceptibility to OMIM:[176885]

About this Structure

1PA1 is a Single protein structure of sequence from Homo sapiens with MG and CL as ligands. Active as Protein-tyrosine-phosphatase, with EC number 3.1.3.48 Full crystallographic information is available from OCA.

Reference

Functional characterization and crystal structure of the C215D mutant of protein-tyrosine phosphatase-1B., Romsicki Y, Scapin G, Beaulieu-Audy V, Patel S, Becker JW, Kennedy BP, Asante-Appiah E, J Biol Chem. 2003 Aug 1;278(31):29009-15. Epub 2003 May 13. PMID:12748196

Page seeded by OCA on Fri Feb 15 16:39:32 2008

Proteopedia Page Contributors and Editors (what is this?)

OCA