The Biosynthesis of Long‐Chain Fatty Acids. Incorporation of Radioactivity from Stereospecifically Tritiated Malonyl Thiol Esters, and the Stereochemistry of the Acetyl‐CoA Carboxylase Reaction

1 Fatty acid synthetase was purified to homogeneity from bakers' yeast. 2 (2 S ,3 R )‐[3‐ 3 H 1 ]Malic acid was prepared by incubation of fumarate with fumarase in tritiated water. 3 [2‐ 3 H 1 ,3‐ 3 H 1 ]Fumarate was synthesised via a published procedure and used for the preparation of (2 S ,3 S )‐[2‐ 3 H 1 ,3‐ 3 H 1 ]malate by incubation with fumarase in water. 4 The above malates after being mixed with [U‐ 14 C]malate were converted into the 4‐nitrophenyl hydrogen malates esterified at the C‐4 carboxyl, the C‐1 carboxyl and the 2‐hydroxyl group being protected at intermediate stages as a 1,3‐dioxolanone. 5 The individual 4‐nitrophenyl hydrogen malates were oxidised by zinc permanganate in carefully defined conditions to give 4‐nitrophenyl hydrogen 2 R ‐[U‐ 14 C, 2‐ 3 H 1 ]‐ and 2 S ‐[U‐ 14 C, 2‐ 3 H 1 ]malonates. 6 These 4‐nitrophenyl hydrogen malonates were converted to the corresponding malonyl thiol esters by transesterification with coenzyme A or N ‐acetylcysteamine under conditions of minimum tritium exchange and racemization. 7 The malonyl thiol esters were immediately incubated with purified fatty acid synthetase and the cofactors necessary for the biosynthesis of fatty acids. 8 The fatty acids, mainly palmitate and stearate, formed in these incubations were extracted and the 3 H/ 14 C ratio determined. Individual acids were recrystallised with carrier material or, after methylation, were separated by gas‐liquid chromatography and the isotope ratio again determined. 9 In this way it was shown that palmitate or stearate derived from the 2 S ‐[U‐ 14 C, 2‐ 3 H 1 ] malonyl thiol ester retained 51% of the original tritium of the substrate whereas the acids derived from the 2 R ‐[U‐ 14 C, 2‐ 3 H 1 ]malonyl thiolester retained only 23% of the original tritium. 2 RS ‐[U‐ 14 C, 2‐ 3 H 1 ] substrates gave an intermediate result. 10 From a comparison of these results with those obtained previously using chiral acetate substrates it is deduced that carboxylation of acetyl‐CoA catalysed by the chicken liver acetyl‐CoA carboxylase occurs with retention of configuration at C‐2. The results are shown to be in quantitative agreement with the theory, deduced from experiments with chiral acetates, that partial exchange of carbon‐bound hydrogen occurs on the synthetase at some stage between malonate and fatty acid subsequent to the stereospecific elimination of hydrogen. 11 The stereochemical course of individual reactions in the biosynthesis of fatty acids is discussed.