Mechanistic Study of an Improbable Reaction: Alkene Dehydrogenation by the Δ12 Acetylenase of Crepis alpina

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Abstract
The mechanism by which the fatty acid acetylenase of Crepis alpina catalyzes crepenynic acid ((9Z)-octadeca-9-en-12-ynoic acid) production from linoleic acid has been probed through the use of kinetic isotope effect (KIE) measurements. This was accomplished by incubating appropriate mixtures of regiospecifically deuterated isotopomers with a strain of Saccharomyces cerevisiae expressing a functional acetylenase. LC/MS analysis of crepenynic acid obtained in these experiments showed that the oxidation of linoleate occurs in two discrete steps, since the cleavage of the C12−H bond is very sensitive to isotopic substitution (kH/kD = 14.6 ± 3.0) while a minimal isotope effect (kH/kD = 1.25 ± 0.08) was observed for the C13−H bond breaking step. These data suggest that crepenynic acid is produced via initial H-atom abstraction at C12 of a linoleoyl substrate. The relationship between the mechanism of enzymatic acetylenation and epoxidation is discussed.

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