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, 3H1]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 acetyl-CoA 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 H1, 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.

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Sedgwick B, Cornforth JW

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