Acetoacetyl-CoA reductase activity of lactating bovine mammary fatty acid synthase.
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 Km of 5 microM acetoacetyl-CoA and a Vmax of about 4 mumol of NADPH oxidized min-1 mg-1. In contrast, the Km for the model compound, acetoacetyl pantetheine was 820 microM 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 2-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 CoA.
D000090:Acetoacetates / Q000037:antagonists & inhibitors / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000031:analogs & derivatives / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000037:antagonists & inhibitors / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002645:Chickens; D003065:Coenzyme A / Q000031:analogs & derivatives / Q000378:metabolism; D010856:Columbidae; D003437:Crotonates / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000378:metabolism*; D005260:Female; D005777:Geese; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000201:enzymology*; D010084:Oxidation-Reduction; D011247:Pregnancy; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000090:Acetoacetates; D000214:Acyl Coenzyme A; D003437:Crotonates; D013439:Sulfhydryl Reagents; C010667:acetoacetyl CoA; C030372:3-hydroxybutyryl-coenzyme A; D000105:Acetyl Coenzyme A; C010701:crotonyl-coenzyme A; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase; D064429:Fatty Acid Synthases; D003065:Coenzyme A