Studies on the Metabolism of Unsaturated Fatty Acids. VI. Stereochemical Studies of the Reaction catalyzed by cis-2-Enoyl-Coenzyme A Reductase of Escherichia coli

Abstract

The stereochemical mechanism of the reduction of cis-2-octenoyl-coenzyme A (-CoA) catalyzed by cis-2-enoyl-CoA reductase from E. coli was studied. cis-2-[7, 8-²H₅] Octenoyl-CoA was incubated with partially purified cis-2-enoyl-CoA reductase in the presence of 4R or 4S-[4-²H₁] nicotinamide adenine dinucleotide phosphate (NADPH). The octenoyl-CoA was also incubated with the enzyme in the presence of NADPH in ²H₂O. Octanoic acids synthesized were isolated and analyzed by gas chromatography-mass spectrometry (GC-MS) to examine the localization of deuterium atoms in the molecule. The octanoic acids isolated from the incubation mixtures were converted to their CoA esters, and then they were dehydrogenated by the action of acyl-CoA oxidase, which had previously been shown to catalyze the anti-elimination of the pro-2R and pro-3R hydrogens of acyl-CoA. The resulting products, trans-2-octenoyl-CoAs, were converted to methyl esters and their deuterium contents at the C-2 or C-3 position were also analyzed by GC-MS. The results suggested the following stereochemical features of the reaction catalyzed by cis-2-enoyl-CoA reductase. 1) pro-4R Hydrogen of NADPH was incorporated into the C-3 position of octenoyl-CoA. 2) Hydrogen from the medium was introduced into the C-2 position of octenoyl-CoA. 3) The reduction occurred by an anti-addition of hydrogen via a 2-Si, 3-Re attack on the cis-double bond.