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

Abstract

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...

Investigation of Fatty Acid Elongation and Desaturation Steps in Fusarium lateritium by Quantitative Two-dimensional Deuterium NMR Spectroscopy in Chiral Oriented Media

“…Because it is observed for the pro-R position of the even CH 2 groups that the participation of water clearly increases (a ij ϭ 0.72; see Equation 16), whereas those of the H-6,6 and H-1 of glucose strongly decrease (0.08; see Equation 17, and 0.01; see Equation 18, respectively), it can be concluded that a major exchange of hydrogen atoms from the CH 3 of acetate with the medium water during chain elaboration has occurred. Thus, post-malonate exchange (10,11), taking place after the formation of malonyl-CoA, is found to strongly influence the hydrogen linkage in the pro-R position of even sites. This process has been described as very variable (11,12,39,40).…”

“…1). However, the isotope redistribution coefficients from acetate will be diminished if post-malonate exchange with medium occurs (10,11). At odd sites (derived from the CϭO of acetate), the hydrogen atoms are introduced by the 3-oxoacyl-CoA reductase and the enoyl reductase, both linked to glucose via the NAD(P)H pool (Fig.…”

Biosynthesis of elsinochromes C and D. Pattern of acetate incorporation determined by 13C and 2H nmr

“…It is noteworthy that the C-1, C-2, C-6, and C-9 methine groups contained no deuterium although also derived from the methyl group of acetate. Acetyl coenzyme A -malonyl coenzyme A interconversion (23) can account for loss of deuterium at the acetyl groups but the complete removal of deuterium at other sites suggests facile exchange between the protons of water and the deuterium at activated methylene groups in malonyl coenzyme A (24,25) or in the polyketide chain bound to the enzyme surface. The presence of deuterium at the methyl groups of 6 but not elsewhere was also confirmed by the 'H spectrum, in which signals (intensity I,,) arising from deuteriums bonded to 13C of the methyl groups were well separated by 13C-'H coupling from the corresponding signal (intensity I,,) of 'H bonded to 12C.…”

Sir John Cornforth Ac Cbe Frs: His Biosynthetic Work

“…Cornforth then turned 43 his attention to the formation of the mevalonic acid precursor, 3-hydroxy-3-methylglutaryl co-enzyme A and to the chain elongation steps (49 → 52) of fatty acid biosynthesis 44,45 . For this he used the enantiomers of malonyl co-enzyme A 50 which, in fatty acid biosynthesis, is formed by the carboxylation of acetyl co-enzyme A.…”