Mechanistic study of an improbable reaction: alkene dehydrogenation by the delta12 acetylenase of Crepis alpina.
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 (k(H)/k(D) = 14.6 +/- 3.0) while a minimal isotope effect (k(H)/k(D) = 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.
Reed
Darwin W
DW
NRC Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9.
Polichuk
Devin R
DR
Buist
Peter H
PH
Ambrose
Stephen J
SJ
Sasata
Robert J
RJ
Savile
Christopher K
CK
Ross
Andrew R S
AR
Covello
Patrick S
PS
D000475:Alkenes / Q000737:chemistry / Q000378:metabolism*; D000480:Alkynes / Q000737:chemistry / Q000378:metabolism; D027468:Crepis / Q000201:enzymology*; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000378:metabolism*; D006865:Hydrogenation; D007700:Kinetics; D008041:Linoleic Acids / Q000737:chemistry / Q000378:metabolism
D000475:Alkenes; D000480:Alkynes; D008041:Linoleic Acids; D044943:Fatty Acid Desaturases; C111962:acetylenase