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DNA inhibits catalysis by the carboxyltransferase subunit of acetyl-CoA carboxylase: Implications for active site communication

Division of Biochemistry and Molecular Biology, Louisiana State University, Baton Rouge, Louisiana 70803, USA

(RECEIVED August 20, 2007; FINAL REVISION October 9, 2007; ACCEPTED October 18, 2007)

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in the synthesis of long-chain fatty acids. The crystal structure of the Escherichia coli carboxyltransferase component of ACC revealed an ₂β₂ subunit composition with two active sites and, most importantly, a unique zinc domain in each β pair that is absent in the eukaryotic enzyme. We show here that carboxyltransferase binds DNA. Half-maximal saturation of different single-stranded or double-stranded DNA constructs is seen at 0.5–1.0 µM, and binding is cooperative and nonspecific. The substrates (malonyl-CoA and biocytin) inhibit DNA:carboxyltransferase complex formation. More significantly, single-stranded DNA, double-stranded DNA, and heparin inhibit the reaction catalyzed by carboxyltransferase, with single-stranded DNA and heparin acting as competitive inhibitors. However, double-inhibition experiments revealed that both DNA and heparin can bind the enzyme in the presence of a bisubstrate analog (BiSA), and the binding of BiSA has a very weak synergistic effect on the binding of the second inhibitor (DNA or heparin) and vice versa. In contrast, DNA and heparin can also bind to the enzyme simultaneously, but the binding of either molecule has a strong synergistic effect on binding of the other. An important mechanistic implication of these observations is that the dual active sites of ACC are functionally connected.

Keywords: acetyl-CoA carboxylase; carboxyltransferase; biotin; zinc finger; DNA binding

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