Biotin Protein Ligase from Saccharomyces cerevisiae
| Autor: | John C. Wallace, Steven W. Polyak, Peter J. Brautigan, Anne Chapman-Smith |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
chemistry.chemical_classification
DNA ligase Protease biology medicine.diagnostic_test medicine.medical_treatment Proteolysis Saccharomyces cerevisiae Active site Cell Biology biology.organism_classification Biochemistry Molecular biology chemistry.chemical_compound Enzyme chemistry Biotin-protein ligase activity Biotin biology.protein medicine Molecular Biology |
| Zdroj: | Journal of Biological Chemistry. 274:32847-32854 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.274.46.32847 |
| Popis: | Catalytically active biotin protein ligase fromSaccharomyces cerevisiae (EC 6.3.4.15) was overexpressed inEscherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. colistrains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function. |
| Databáze: | OpenAIRE |
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