1PA1

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

Summary for 1PA1

• Entry DOI: 10.2210/pdb1pa1/pdb
• Descriptor: Protein-tyrosine phosphatase, non-receptor type 1, MAGNESIUM ION, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: phosphatase, catalytic loop, active-site mutant, hydrolase
• Biological source: Homo sapiens (human)
• Cellular location: Endoplasmic reticulum membrane; Peripheral membrane protein; Cytoplasmic side: P18031
• Total number of polymer chains: 1
• Total formula weight: 36511.44

Authors

Romsicki, Y.,Scapin, G.,Beaulieu-Audy, V.,Patel, S.B.,Becker, J.W.,Kennedy, B.,Asante-Appiah, E. (deposition date: 2003-05-13, release date: 2003-08-05, Last modification date: 2023-08-16)

Primary citation

Romsicki, Y.,Scapin, G.,Beaulieu-Audy, V.,Patel, S.,Becker, J.W.,Kennedy, B.P.,Asante-Appiah, E.
Functional characterization and crystal structure of the C215D mutant of protein-tyrosine phosphatase-1B
J.Biol.Chem., 278:29009-29015, 2003

PubMed 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.

PubMed: 12748196
DOI: 10.1074/jbc.M303817200

Experimental method

X-RAY DIFFRACTION (1.6 Å)