Functional characterization and crystal structure of the C215D mutant of protein-tyrosine phosphatase-1B

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 Angstrom. The mutant enzyme displayed maximal catalytic activity at pH similar to4.5, which is significantly lower than the pH optimum of 6 for wildtype PTP-1B. Although both forms of the enzyme exhibited identical K-m values for hydrolysis of p-nitrophenyl phosphate at pH 4.5 and 6, the k(cat) values of C215D were similar to70- and similar to7000-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 Asp(215) in catalysis. Increasing solvent microviscosity with sucrose (up to 40% (w/v)) caused a significant decrease in k(cat)/K-m 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.