Inhibition of acetyl-CoA carboxylase isoforms by pyridoxal phosphate

Abstract

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 K i 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 K i 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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.