Inactivation of chicken liver fatty acid synthetase by malonyl coenzyme A. Effects of acetyl coenzyme A and nicotinamide adenine dinucleotide phosphate

Abstract

Chicken liver fatty acid synthetase complex is irreversibly inactivated by one of the substrates, malonyl-CoA. Acetyl-CoA has a dual role. At concentrations less than or comparable to those of malonyl-CoA, the rate of inactivation is enhanced, whereas at acetyl-CoA/malonyl-CoA ratios greater than 2, the rte of inactivation is slowed down. NADP+ at low concentrations (25 microM) affords considerable protection against malonyl-CoA mediated inactivation whereas NAD+ even at 1.0 mM concentration has no effect. The inactivation process does not lead to the dissociation of the enzyme complex and is accompanied by subtle conformational changes as measured by circular dichroism measurements. Of all the model partial reactions, decarboxylation of malonyl-CoA and the condensation--CO2 exchange are the only reactions which are not catalyzed by the modified species. The process of inactivation is accompanied by enhanced covalent binding of malonyl groups such that approximately 6 mol of the acyl group is bound per mol of the enzyme at complete inactivation. The available evidence suggests that the inactivation of the enzyme results from the binding of malonyl group(s) at or near the condensing site of the enzyme.

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: reduced nicotinamide adenine dinucleotide phosphate binding and formation and reduction of acetoacetyl-enzyme

“…For both traces 2 and 3, the calculated curves are coincidental with the experimental trace. As expected, if MalCoA is incubated with the enzyme for long times (> 15 min), the enzyme is signifi- cantly inhibited (Kumar & Srinivasan, 1981). If the enzyme is incubated with AcCoA, 10 mM acetyl phosphate, and about 200 units/mL of phosphotransacetylase for 3-62 min and mixed with 100 µ MalCoA and 0.7 µ NADPH, trace 4 is obtained.…”

“…Preincubation of MalCoA-enzyme with phosphotransacetylase does not produce such a dramatic effect, although a substantial fraction of the MalCoA-enzyme mixture reacts slowly with NADPH. This experiment is flawed by the fact that AcCoA is produced by phosphotransacetylase during the incubation and by the complex interactions of MalCoA with the enzyme (Kumar & Srinivasan, 1981). However, malonyl groups do not appear to block the formation of acetoacetyl by the enzyme as effectively as acetyl groups.…”

Investigation of NADPH and acyl-binding sites on avian fatty acid synthase

“…The hydroxyl sites, if not the result of sulfur to oxygen transfers that are not catalytically significant, are more likely to be the thioesterase sites, which bind the active serine reagents tosyl fluoride and diisopropyl fluorophosphate (Kumar, 1975;Lin & Smith, 1978). They also may be related to the hydroxylamine-insensitive sites implicated in malonyl-CoA inhibition of the enzyme (Kumar & Srinivasan, 1981). Our results indicate that the binding to the hydroxylamine-insensitive sites (presumably hydroxyl sites) is most likely subsequent to binding to the thiol sites.…”

Inactivation of 3‐Oxoacyl Synthetase Activity of Pigeon Liver Fatty Acid Synthetase by S‐(4‐Bromo‐2,3‐dioxobutyl)‐coenzyme A

“…The increase in the rate of dibromopropanone inhibition of chicken liver fatty acid synthetase in the presence of high concentrations of malonyl-CoA is probably due to the inhibitory effect of malonyl-CoA itself. It has recently been shown [26] that chicken liver fatty acid synthetase is irreversibly inactivated by one of its substrates, malonyl-CoA. In this respect, chicken liver enzyme is different from the pigeon liver enzyme.…”