1pa1
Crystal structure of the C215D mutant of protein tyrosine phosphatase 1B
Structural highlights
FunctionPTN1_HUMAN Tyrosine-protein phosphatase which acts as a regulator of endoplasmic reticulum unfolded protein response. Mediates dephosphorylation of EIF2AK3/PERK; inactivating the protein kinase activity of EIF2AK3/PERK. May play an important role in CKII- and p60c-src-induced signal transduction cascades. May regulate the EFNA5-EPHA3 signaling pathway which modulates cell reorganization and cell-cell repulsion.[1] [2] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedWe 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. 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[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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