Brownsey, Roger W.

Mammalian acetyl-CoA carboxylase (ACC) isoforms, ACC-1 and ACC-2, catalyze the formation of malonyl-CoA, a substrate for fatty acid synthesis and fatty acyl chain elongation, and a potent inhibitor of beta-oxidation. Based on the phenotype of knockout mice, ACC contributes significantly to overall body energy metabolism and is a potential drug target for the treatment of obesity and type II diabetes. Highly-purified ACC-1 undergoes a dramatic increase in mass following treatment with allosteric activators such as citrate, apparently due to linear polymerization of ACC dimers. The current studies were undertaken to explore (a) the extent to which ACC-2 undergoes polymerization and (b) to investigate whether other cellular proteins associate with and perhaps regulate the polymerization process. Size exclusion chromatography and sucrose gradient sedimentation studies show that ACC-2 undergoes very modest changes in molecular size upon allosteric activation, although the extent of polymerization of ACC-2 is enhanced in the presence of ACC-1. Isolation of the largest molecular forms of ACC, followed by mass spectrometry analysis, reveals that ACC "polymers" are multi-protein complexes. The proteins most reliably detected in ACC polymer fractions include tubulin, actin, fatty acid synthase, and heat shock proteins. The significance of the additional components of ACC complexes is evaluated from effects on the kinetics of the ACC reaction and of the polymerization process, as well as by cellular colocalization using fluorescence microscopy and co-immunoprecipitation studies. [ABSTRACT FROM AUTHOR]

Mammalian isoforms of acetyl-CoA carboxylase (ACC-1 and ACC-2) play important roles in synthesis, elongation, and oxidation of long-chain fatty acids, and the possible significance of ACC in the development of obesity has led to interest in the development of inhibitors. Here, we demonstrate that pyridoxal phosphate (PLP) is a linear and reversible inhibitor of ACC-1 and ACC-2. ACC from rat liver and white adipose tissue (largely ACC-1) exhibited an IC50 of ∼200 μM, whereas ACC-2 from heart or skeletal muscle exhibited an IC50 exceeding 500 μM. ACC from rat liver was equally sensitive to PLP following extensive purification by avidin affinity chromatography. When added before citrate, PLP inhibited ACC with a Ki of ∼100 μM, reducing maximal activity >90% and increasing the Ka for citrate ∼5-fold but having little effect on substrate Km values. Pre-treatment with citrate increased the apparent Ki for ACC inhibition by PLP by ∼4-fold. Inhibition of ACC was reversed by removal of PLP, either by washing or by reaction with hydroxylamine or amino-oxyacetate. ACC was irreversibly inhibited and radiolabeled, to a stoichiometry of ∼0.4 mol[H]/mol subunit, in the presence of PLP plus [³H]borohydride. Studies with structurally related compounds demonstrated that the reactive aldehyde and negatively charged substituents of PLP contribute importantly to ACC inhibition. The studies reported here suggest a rationale to develop ACC inhibitors that are not structurally related to the substrates or products of the reaction and an approach to probe the citrate-binding site of the enzyme. [ABSTRACT FROM AUTHOR]