Inhibition of acetyl-CoA carboxylase isoforms by pyridoxal phosphate.
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 approximately 200 microm, whereas ACC-2 from heart or skeletal muscle exhibited an IC50 exceeding 500 microm. 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 approximately 100 microm, reducing maximal activity >90% and increasing the Ka for citrate approximately 5-fold but having little effect on substrate Km values. Pre-treatment with citrate increased the apparent Ki for ACC inhibition by PLP by approximately 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 approximately 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.
Lee
Weissy M
WM
Department of Biochemistry and Molecular Biology & Life Sciences Institute, Diabetes Research Group, The University of British Columbia, Vancouver, British Columbia, Canada.
Elliott
Jason E
JE
Brownsey
Roger W
RW
D000103:Acetyl-CoA Carboxylase / Q000037:antagonists & inhibitors*; D000818:Animals; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D008297:Male; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011732:Pyridoxal Phosphate; D000103:Acetyl-CoA Carboxylase