title: Complex formation and regulation of Escherichia coli acetyl-CoA carboxylase.
abstract: Acetyl-CoA carboxylase is a biotin-dependent enzyme that catalyzes the regulated step in fatty acid synthesis. The bacterial form has three separate components: biotin carboxylase, biotin carboxyl carrier protein (BCCP), and carboxyltransferase. Catalysis by acetyl-CoA carboxylase proceeds via two half-reactions. In the first half-reaction, biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin, which is covalently attached to BCCP, to form carboxybiotin. In the second half-reaction, carboxyltransferase transfers the carboxyl group from carboxybiotin to acetyl-CoA to form malonyl-CoA. All biotin-dependent carboxylases are proposed to have a two-site ping-pong mechanism in which the carboxylase and transferase activities are separate and do not interact. This posits two hypotheses: either biotin carboxylase and BCCP undergo the first half-reaction, BCCP dissociates, and then BCCP binds to carboxyltransferase, or all three constituents form an enzyme complex. To determine which hypothesis is correct, a steady-state enzyme kinetic analysis of Escherichia coli acetyl-CoA carboxylase was conducted. The results indicated the two active sites of acetyl-CoA carboxylase interact. Both in vitro and in vivo pull-down assays demonstrated that the three components of E. coli acetyl-CoA carboxylase form a multimeric complex and that complex formation is unaffected by acetyl-CoA, AMPPNP, and mRNA encoding carboxyltransferase. The implications of these findings for the regulation of acetyl-CoA carboxylase and fatty acid biosynthesis are discussed.
authors.affiliation: Division of Biochemistry and Molecular Biology, Louisiana State University , Baton Rouge, Louisiana 70803, United States.
authors.forename: Tyler C
authors.initials: TC
authors.lastname: Broussard
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Price
authors.forename: Susan M
authors.initials: SM
authors.lastname: Laborde
authors.forename: Grover L
authors.initials: GL
authors.lastname: Waldrop
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019878:Carboxyl and Carbamoyl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D019878:Carboxyl and Carbamoyl Transferases; D054889:Fatty Acid Synthase, Type II; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein