Intermediates of fatty acid synthesis: Sites of binding to the pigeon liver fatty acid synthetase

Abstract

The pigeon-liver fatty acid synthetase complex contains three acetyl- and two malonyl-binding sites. One of these sites (B 1) appears to be a hydroxyl-containing compound. Acetyl and malonyl groups (transferred from their CoA esters) are bound to this site in increased amounts when the sulfhydryl sites that bind acyl groups to the enzyme are blocked with iodoacetamide. This result suggests that B 1 is a primary or loading site for acetyl and malonyl groups. Competitive inhibition studies have shown that acetyl and malonyl groups compete for this site, thereby suggesting that the fatty acid synthetase may have a single loading site (B 1) for these groups. Similarly, acetyl and malonyl groups bind competitively to the 4′-phosphopantetheine (A 2) prosthetic group of the enzyme. A third site (B 2) binds acetyl, but not malonyl, groups to the enzyme. The acetoacetyl moiety is bound to the 4′-phosphopantetheine (A 2) site of the enzyme. The formation of the acetoacetyl group on this site has been shown to be deendent on the availability of the B 2 site for the binding of the acetyl group. A reduction of acetyl binding to this site, by selective iodoacetamide inhibition resulted in a parallel reduction in the formation of acetoacetyl-enzyme. Incubation of the enzyme with malonyl-CoA and hexanoyl-CoA yields β-ketooctanoyl-enzyme. The β-ketooctanoyl moiety is bound to the enzyme as a thioester of 4′-phosphopantetheine. Reduction of β-ketooctanoyl-enzyme in the presence of NADPH yields octanoyl-enzyme as a product. The octanoyl group is bound to the enzyme through thioester bonding at both the A 2 (4′-phosphopantetheine) and B 2 sites. The location of intermediates on specific prosthetic groups of the enzyme is utilized to deduce both the order of the intermediate reactions and the role played by each prosthetic group during fatty acid synthesis.