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1. Fatty acid synthetase was purified to homogeneity from bakers' yeast. (2S,3R)-[3-3H~]Malic acid was prepared by incubation of fumarate with fumarase in tritiated water.3. [2-3H1 ,3-3H~]Fumarate was synthesised via a published procedure and used for the preparation of (2S,3S)-[2-3H~,3-3Hl]malate by incubation with fumarase in water.4. The above malates after being mixed with [U-14C]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 2R-[U-14C, 2-3H1]-and 2S-[U-I4C, 2-3Hl]malonates.6. These 4-nitrophenyl hydrogen malonates were converted to the corresponding malonyl thiol esters by transesterification with coenzyme A or A'-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 3H/14C 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 2S-[U-14C, 2 -3 H~] malonyl thiol ester retained 51 % of the original tritium of the substrate whereas the acids derived from the 2R-[U-14C, 2-3H1]malonyl thiolester retained only 23 % of the original tritium. 2RS-[U-14C, 2-3H1] 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.In the foregoing paper [l] it was shown that the formation of fatty acids from chiral acetates. The fatty acid synthetase from chicken liver and bakers' discrimination between the R and S enantiomers in yeast possessed an overall stereospecificity in the these experiments was small because the small hydrogen isotope effect, operative in the acetyl-CoA carboxylase reaction, led to nearly equal proportions of tritiated malonyl-CoA species having R and Schirality. A partial, and therefore non-specific, exchange of hydrogen catalysed by the synthetase was also obAbbreviations. GLC. gas-liquid chromatography; TLC, thinlayer chrom...

A known catabolic pathway of S-methyl-L-methionine in higher plants: donation of a methyl group to L-homocysteine resulting in the production of two molecules of L-methionine, is subjected to stereochemical studies. The two, diastereoisomeric (2-2H, methyl-' 3C)-S-methyl-~-methionines are synthesized and utilised in transmethylation reactions with L-homocysteine as the acceptor and an enzyme preparation from jack beans as a catalyst. The resulting, variously labelled methionine species are converted into butyl esters of the N-trifluoroacetylated derivatives and, as such, subjected to g.1.c. combined with mass spectrometry in two ionisation modes. Experimentally determined parameters such as mass peak intensities, isotopic enrichment factors, diastereoisomeric purities, and protein-derived methionine, are utilised for calculating the stereoselectivity in the enzyme transfer of the diastereoisotopic methyl groups from S-methyl -L-methionine to L-homocysteine. Together, the independent results from the two series of diastereoisomers reveal an enzymic preference of the pro-(R) -* For preliminary accounts of this work,

The absolute configuration of methylethylpropylsulphonium ion has been established by chemical correlation with lactic acid through an intermediate, stereochemically identified by X-ray analysis.

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