1pa1: Difference between revisions
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==Overview== | ==Overview== | ||
We have characterized the C215D active-site mutant of protein-tyrosine | 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== | ==Disease== | ||
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[[Category: Asante-Appiah, E.]] | [[Category: Asante-Appiah, E.]] | ||
[[Category: Beaulieu-Audy, V.]] | [[Category: Beaulieu-Audy, V.]] | ||
[[Category: Becker, J | [[Category: Becker, J W.]] | ||
[[Category: Kennedy, B.]] | [[Category: Kennedy, B.]] | ||
[[Category: Patel, S | [[Category: Patel, S B.]] | ||
[[Category: Romsicki, Y.]] | [[Category: Romsicki, Y.]] | ||
[[Category: Scapin, G.]] | [[Category: Scapin, G.]] | ||
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[[Category: phosphatase]] | [[Category: phosphatase]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:26:43 2008'' | ||
Revision as of 15:26, 21 February 2008
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Crystal structure of the C215D mutant of protein tyrosine phosphatase 1B
OverviewOverview
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.
DiseaseDisease
Known diseases associated with this structure: Abdominal body fat distribution, modifier of OMIM:[176885], Insulin resistance, susceptibility to OMIM:[176885]
About this StructureAbout this Structure
1PA1 is a Single protein structure of sequence from Homo sapiens with and as ligands. Active as Protein-tyrosine-phosphatase, with EC number 3.1.3.48 Full crystallographic information is available from OCA.
ReferenceReference
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
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