PMID:8631788
| Citation |
Nenortas, E and Beckett, D (1996) Purification and characterization of intact and truncated forms of the Escherichia coli biotin carboxyl carrier subunit of acetyl-CoA carboxylase. J. Biol. Chem. 271:7559-67 |
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| Abstract |
Biotin biosynthesis and retention in Escherichia coli is regulated by the multifunctional protein, BirA. The protein acts as both the transcriptional repressor of the biotin biosynthetic operon and as a ligase for covalent attachment of biotin to a unique lysine residue of the acetyl-CoA carboxylase. Biotinyl-5'-AMP is the activated intermediate for the ligase reaction and the allosteric effector for DNA binding. We have purified and characterized apoBCCP and a truncated form containing the COOH-terminal 87 residues (apoBCCP87). Molecular masses of the proteins measured using matrix-assisted laser desorption ionization time-of-flight mass spectrometry conformed to the expected values. The assembly states of apoBCCP and apoBCCP87 were determined using sedimentation equilibrium ultracentrifugation. Nearly quantitative enzymatic transfer of biotin from BirA-biotinyl-5'-AMP to the apoBCCP forms was assessed using two methods, mass spectrometric analysis of acceptor proteins after incubation with BirA-bio-5'-AMP and a steady state fluorescence assay. The BirA catalyzed rates of transfer of biotin from bio-5'-AMP to apoBCCP and apoBCCP87 were measured by stopped-flow fluorescence. Kinetic parameters estimated from these measurements indicate that the intact and truncated forms of the acceptor protein are functionally identical. |
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| Keywords |
Acetyl-CoA Carboxylase/chemistry; Acetyl-CoA Carboxylase/isolation & purification; Acetyl-CoA Carboxylase/metabolism; Biotin/biosynthesis; Biotin/metabolism; Carrier Proteins/chemistry; Carrier Proteins/isolation & purification; Carrier Proteins/metabolism; Escherichia coli/metabolism; Fatty Acid Synthetase Complex, Type II; Kinetics; Macromolecular Substances; Mass Spectrometry; Molecular Weight; Peptide Fragments/chemistry; Peptide Fragments/isolation & purification; Protein Conformation; Sequence Deletion; Spectrometry, Fluorescence; Ultracentrifugation |
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