Evidence for distinct ligand-bound conformational states of the multifunctional Escherichia coli repressor of biotin biosynthesis.
The Escherichia coli repressor of biotin biosynthesis (BirA) is a unique transcriptional repressor which catalyzes synthesis of its own corepressor and catalyzes attachment of a cofactor to an essential metabolic enzyme. BirA both catalyzes synthesis of biotinyl-5'-AMP from the substrates ATP and biotin and transfer of the biotin moiety from the adenylate to a lysine residue of a subunit of the acetyl-CoA carboxylase. BirA-bio-5'-AMP, moreover, binds sequence specifically to the biotin operator to repress transcription of the biotin biosynthetic genes. Using a combination of kinetic measurements of binding of the two ligands, biotin and bio-5'-AMP, to BirA as well as proteolytic digestion experiments, we have found evidence for at least three discrete conformational states of BirA. Results of stopped-flow fluorescence measurements of association of both ligands with BirA indicate that the process involves initial formation of a collision complex followed by a slow conformational change. The kinetics of the conformational change are distinct for the two ligands and are the basis for the difference in the thermodynamic stabilities of the two protein-ligand complexes. Different rates of proteolytic digestion of apoBirA and complexes of BirA with the two ligands were also observed. Results of the combined approaches indicate that apoBirA, and the BirA-bio-5'-AMP and BirA-biotin complexes are conformationally distinct.
Xu
Y
Y
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228, USA.
Nenortas
E
E
Beckett
D
D
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors
C082099:5'-AMP-biotin; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D012097:Repressor Proteins; D014157:Transcription Factors; D000249:Adenosine Monophosphate; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli