Steric Course of Deuterium Incorporation from [2-2H2]Manlonyl-CoA into Fatty Acids by Fatty Acid Synthetases1

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Abstract
The steric course of the ertoyl reduction catalyzed by fatty acid synthetase was investigated with the enzymes from bakers' yeast, rat liver and Brevibacrerium ammoniagenes. The non-enzymic hydrogen-deuterium exchange of the methylene group of malonyl-CoA was studied by NMR spectroscopy. The half-life period of the methylene protons was 4.8 mm at 37°C and 12.2 mm at 23°C at p2H 7.5. Deuterium-labeled fatty acids were synthesized by incubating the synthetases with [2-2H2]malonyl-CoA for 8 mm. The deuterium-labeled fatty acids thus produced were extracted and subjected to the action of acyl-CoA oxidase, which had been previously shown to catalyze the anti elimination of the pro–2R and pro-3R hydro gens of acyl-CoA. The resulting products, 2,3-dehydroacyl-CoAs, were methylated and converted to 3-chlorofatty acid methyl esters by addition of hydrogen chloride. The deuterium contents of saturated fatty acids and 3-chlorofatty acids were ana lyzed by gas chromatography-mass spectrometry. The oleic acid produced by the enzyme from B. ammoniagenes was oxidized to nonanoic acid and azelaic acid. The resulting nonanoic acid was also subjected to the action of acyl-CoA oxidase. The deuterium contents of nonanoic acid and trans-2-nonenoic acid were analyzed. The results suggested that fatty acid synthetases from yeast and rat liver incorporated hydrogen from water via a 2-Si attack and the enzyme from B. ammoniagenes incor porated hydrogen via a 2-Re attack during enoyl reduction. The partial racemiza tion of the C-2 position was observed and the magnitude of this racemization was correlated with the deuterium content of synthesized fatty acids. This phenomenon may be attributed to the non-stereospecific hydrogen exchange of the C-2 position of the elongating acyl residue catalyzed by fatty acid synthetases.

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