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

J Biol Chem. 2003 Aug 1;278(31):29009-15. doi: 10.1074/jbc.M303817200. Epub 2003 May 13.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding Sites
  • Catalysis
  • Crystallization
  • Crystallography, X-Ray
  • Humans
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Models, Molecular
  • Molecular Structure
  • Mutagenesis, Site-Directed*
  • Nitrophenols / metabolism
  • Organophosphorus Compounds / metabolism
  • Protein Conformation
  • Protein Tyrosine Phosphatase, Non-Receptor Type 1
  • Protein Tyrosine Phosphatases / chemistry*
  • Protein Tyrosine Phosphatases / genetics
  • Protein Tyrosine Phosphatases / physiology*
  • Structure-Activity Relationship
  • Thermodynamics
  • Viscosity

Substances

  • Nitrophenols
  • Organophosphorus Compounds
  • nitrophenylphosphate
  • PTPN1 protein, human
  • Protein Tyrosine Phosphatase, Non-Receptor Type 1
  • Protein Tyrosine Phosphatases

Associated data

  • PDB/1PA1