Glyceride biosynthesis catalysed by the mitochondrial fraction of rat liver or by the microsoma1 fraction of cat intestinal mucosa is greatly stimulated by the supernatant fraction (6,000,000 x g x min). This stimulation is due to the presence of several factors and evidence is presented that one of these factors is a phosphatidate phosphohydrolase. The catalytic activities of the phosphatidate phosphohydrolases present in the mitochondrial and supernatant fractions were studied using as subxtrates either an aqueous dispersion of phosphatidate or membranebound phosphatidate formed as an intermediate in the biosynthesis of glycerides.The mitochondrial phosphatidate phosphohydrolase has a high activity with aqueous phosphatidate dispersions and a low activity with membrane-bound phosphatidate present as internal or added substrate. I n contrast, the phosphatidate phosphohydrolase of the supernatant fraction has a low activity with aqueous phosphatidate dispersions and a high activity with membranebound phosphatidate.As Mg++ and F-ions are usually included in the assay systems employed for measuring glyceride biosynthesis, the effect of these ions on the particulate and soluble phosphatidate phosphohydrolases was studied to exclude the possibility that the stimulation of glyceride biosynthesis by the phosphohydrolase of the supernatant fraction was due to an artefact. It is suggested that the phosphatidate phosphohydrolase isolated in the supernatant fraction is the major phosphohydrolase activity involved in glyceride biosynthesis.
Dehydration of the (3R)-hydroxyalkyl S-thioester (1) by yeast fatty acid synthetase to give the trans-2-enoyl derivative (2) occurs by means of a synelimination of the elements of water.
1. Acetyl-CoA carboxylase and fatty acid synthetase were purified from chicken liver. Fatty acid synthetase was also purified from bakers' yeast.2. The corresponding CoA thiol esters were prepared enzymically from R-[2H1, 3H~]acetate, S-[2H1, 3Hl]acetate and [3Hl]acetate, using a linked acetate kinase/phosphotransacetylase system. [2-14C]Acetate was added to the tritiated specimens to provide doubly-labelled products, which were purified by column chromatography on DEAE-cellulose.3. The three acetyl-CoA specimens thus obtained were incubated separately in a combined acetylCoA carboxylase/fatty acid synthetase system containing the cofactors necessary for synthesis of long-chain fatty acids.4. The fatty acids formed were extracted into n-pentane, methylated, separated by gas-liquid chromatography and the 3H/14C isotope ratio determined.5. In this manner it was shown, using the chicken liver synthetase, that in palmitic acid a higher proportion of tritium was retained from S-[2-I4C, 2H1, 'Hl]acetyl-CoA than from the R-(2-14C, 2 H ,, 3H1)-labelled thiol ester, the non-chiral (2-14C, 3H1)-labelled substrate giving an intermediate result.6. This result was confirmed using the fatty acid synthetase preparation purified from bakers' yeast in place of the corresponding chicken liver enzyme.7. In a separate experiment it was shown that the intramolecular tritium isotope effect in the carboxylation of [3Hl]acetyl-CoA is normal, but small. The slightly smaller, normal deuterium isotope effect calculated from this was shown to be in good agreement with the value expected, based on the experiments on tritium incorporation from chiral acetyl-CoA.8. It is demonstrated that partial exchange of carbon-bound hydrogen must occur on the synthetase during the enzymic synthesis of fatty acids from malonyl thiol esters and that the extent of this exchange differs in synthetases of different origin.9. A theoretical treatment of the effect of partial hydrogen exchange on the chirality of asymmetrically labelled methylene groups is given.10. The results indicate the existence of an overall stereospecificity in the reactions involved in the de novo biosynthesis of long-chain fatty acids in different cell types. ~~~Abbreviations.
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...
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