PURIFICATION AND CHARACTERIZATION OF INTACT AND TRUNCATED FORMS OF THE ESCHERICHIA-COLI BIOTIN CARBOXYL CARRIER SUBUNIT OF ACETYL-COA CARBOXYLASE

Biotin biosynthesis and retention in Escherichia coli is regulated by the multifunctional protein, BirA. The protein acts as both the transc riptional repressor of the biotin biosynthetic operon and as a ligase for covalent attachment of biotin to a unique lysine residue of the bi otin carboxyl carrier protein (BCCP) subunit of the acetyl-CoA carboxy lase. Biotinyl-5'-AMP is the activated intermediate for the ligase rea ction and the allosteric effector for DNA binding. We have purified an d characterized apoBCCP and a truncated form containing the COOH-termi nal 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 incub ation 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 apoBCCPS7 were measured by stopped-flow fluorescence. Kinetic para meters estimated from these measurements indicate that the intact and truncated forms of the acceptor protein are functionally identical.