Alpha-secondary isotope effects in the lipoxygenase reaction.
Isotope effects for the oxidation of [5,6,8,9,11,12,14,15-3H]arachidonic acid catalyzed by soybean lipoxygenase and by 5-lipoxygenase were measured. This labeling pattern represents substitution at each of the vinylic hydrogens of the substrate. The observed isotope effect for soybean lipoxygenase was 1.16 +/- 0.02 and for 5-lipoxygenase was 1.11 +/- 0.05. These isotope effects are inconsistent with any change in hybridization (sp2 to sp3) at the vinylic carbons prior to or during the rate-determining step and are concluded to be most consistent with the formation of a carbanion-like intermediate or transition state. In contrast, the oxidation of arachidonic acid by Ce(IV), which is thought to proceed via a cation radical intermediate, exhibited at most a small isotope effect (1.02 +/- 0.01). The reduction potential for the cation radical formed from arachidonic acid in this reaction is estimated to be 2.7 V vs NHE by comparison of the rates of oxidation of arachidonic acid and cyclohexene by Ce(IV). This is similar to the potential for the cation radical of 2-butene. No isotope effect (1.00 +/- 0.03) was observed in the 5-lipoxygenase reaction for conversion of the initially formed product 5-hydroperoxyeicosatetraenoic acid to the epoxide leukotriene A4. From this it is concluded that there is little carbon-oxygen bond formation prior to or during the rate-determining step for epoxide formation.
Wiseman
J S
JS
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008084:Lipoxygenase / Q000302:isolation & purification; D010084:Oxidation-Reduction; D013025:Glycine max / Q000201:enzymology; D014316:Tritium
D001095:Arachidonic Acids; D007527:Isoenzymes; D014316:Tritium; D016718:Arachidonic Acid; D008084:Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase