The C‐terminal domain of biotin protein ligase from E. coli is required for catalytic activity

摘要: Biotin protein ligase of Escherichia coli, the BirA protein, catalyses covalent attachment biotin prosthetic group to a specific lysine carboxyl carrier (BCCP) subunit acetyl-CoA carboxylase. also functions repress biosynthetic operon and synthesizes its own corepressor, biotinyl-5′-AMP, catalytic intermediate in biotinylation reaction. We have previously identified two charge substitution mutants BCCP, E119K, E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis investigate residues may interact with E119 or E147 BCCP. None complementary mutations at selected restored activity wild-type levels when assayed our BCCP mutant substrates. However, variant, which K277 C-terminal domain was substituted Glu, had significantly higher E119K than did No function has been for domain, is distinct from central thought contain ATP binding site known site. Kinetic analysis several purified enzymes indicated single amino acid within (R317E) located some distance presumptive resulted 25-fold decrease affinity ATP. Our data indicate essential enzyme contributes interaction substrate, domain.