Acetoacetyl-CoA reductase activity of lactating bovine mammary fatty acid synthase.

Abstract

Fatty acid synthase, purified from lactating bovine mammary gland, utilizes coenzyme A esters of acetoacetic, 3-hydroxybutyric, and crotonic acids as substrates for its partial reactions at micromolar concentrations.The NADPH:acetoacetyl-CoA reductase had a K,,, of 5 PM acetoacetyl-CoA and a V,, of about 4 pmol of NADPH oxidized min" mg".In contrast, the X,,, for the model compound, acetoacetyl pantetheine was 820 PM and that of S-acetoacetyl-N-acetylcysteamine was over 40 mM.The reduction of acetoacetyl-CoA was observed with the enzyme from rat tissues also but not with those from avian tissues or yeast.With the bovine mammary enzyme, the reaction was found to oxidize 2 mol of NADPH for every mol of acetoacetyl-CoA consumed.Butyrate was the major product of reduction.The reductase activity was susceptible to inhibition by several sulfhydryl reagents; it was lost when the synthase was dissociated into one-half molecular weight subunits or when the incubation mixture was depleted of CoA.It was competitively inhibited by acetyl-coA, butyryl-CoA, methylmalonyl-CoA, and Z-methylcrotonyl-CoA.These results as well as its use as a primer in fatty acid synthesis by the enzyme suggest that the acetoacetyl group from acetoacetyl-CoA is transferred to the enzyme, presumably to its 4'-phosphopantheine prosthetic group.The acyl group is then expected to remain attached to the enzyme while it is reduced, dehydrated, and reduced again to form a butyryl group which can either undergo chain elongation, if malonyl-CoA is present, or be released from the enzyme by hydrolysis or transfer to free COG.Fatty acid synthase in eukaryotes is a multifunctional enzyme complex, classified as Type 1 (l), which catalyzes the seven or more individual reactions involved in the sequential addition of two carbon units, derived from malonyl-CoA, to a primer acetyl-coA or butyryl-CoA (1, 2 ) .Currently, there is general agreement that the enzyme consists of an aggregate of two polypeptide chains which are identical in the enzyme