The Distinctive Regulatory Mechanisms of Bacterial Acetyl-CoA Carboxylase

Metabolic Regulation is a complex system used to control cellular metabolism in response to conditions in the cell’s environment. For most enzymes, the cell can rely upon a minimal amount of regulation; however, critical enzymes, such as acetyl-CoA carboxylase, must be regulated at multiple levels. Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In bacteria, acetyl-CoA carboxylase forms a complex of three subunits–biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase–which catalyze the carboxylation of acetyl-CoA to form malonyl-CoA via two half-reactions. In the first half-reaction, biotin covalently linked to biotin carboxyl carrier protein is carboxylated by biotin carboxylase. Carboxyltransferase catalyzes the second half-reaction where the carboxyl group is transferred from carboxybiotin to acetyl-CoA, forming malonyl-CoA. As acetyl-CoA carboxylase is a critical enzyme in cell growth, it is subject to multiple forms of regulation. This dissertation describes two distinct mechanisms of regulation of acetyl-CoA carboxylase. Previous reports showed that the downstream product of fatty acid synthesis, palmitoyl-acyl carrier protein, inhibits catalysis by acetyl-CoA carboxylase via negative feedback regulation. This dissertation reports the acetyl-CoA carboxylase complex was found to exhibit a pronounced hysteresis when inhibited by palmitoyl-acyl carrier protein. Alternatively, palmitoyl-acyl carrier protein does not inhibit either half–reaction. Structure-function studies of palmitoyl-acyl carrier protein demonstrate the inhibitory moiety is the cofactor pantothenic acid. This dissertation also reports that the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase regulates catalysis. The biotin carboxyl carrier protein in E. coli is composed of two domains: the N-terminal domain, and the C-terminal domain. The C-terminal domain contains the biotin cofactor, and acts as a substrate, while the function of the N-terminal domain was unknown. In vivopull-down assays demonstrated the N-terminal domain is not required for formation of the catalytic complex, whereasin vitroanalyses demonstrated that the N-terminal domain binds biotin. Thus, it is likely the role of the N-terminal domain of biotin carboxyl carrier protein in bacterial acetyl-CoA carboxylase is to bind and stabilize the carboxybiotin intermediate during catalysis