Oxygenation of monounsaturated fatty acids by soybean lipoxygenase-1: evidence for transient hydroperoxide formation.
Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Previous work from our laboratories has demonstrated that SBLO-1 will also catalyze the oxygenation of monounsaturated acids (Clapp, C. H., Senchak, S. E., Stover, T. J., Potter, T. C., Findeis, P. M., and Novak, M. J. (2001) Soybean Lipoxygenase-Mediated Oxygenation of Monounsaturated Fatty Acids to Enones, J. Am. Chem. Soc. 123, 747-748). Interestingly, the products are alpha,beta-unsaturated ketones rather than the expected allylic hydroperoxides. In the present work, we provide evidence that the monoolefin substrates are initially converted to allylic hydroperoxides, which are subsequently converted to the enone products. The hydroperoxide intermediates can be trapped by reduction to the corresponding allylic alcohols with glutathione peroxidase plus glutathione or with SnCl2. Under some conditions, the hydroperoxide intermediates accumulate and can be detected by HPLC and peroxide assays. Kinetics measurements at low concentrations of [1-14C]-9(Z)-octadecenoic acid indicate that oxygenation of this substrate at 25 degrees C, pH 9.0 occurs with kcat/Km = 1.6 (+/-0.1) x 10(2) M-1 s-1, which is about 105 lower than kcat/Km for oxygenation of 9(Z),12(Z)-octadecadienoic acid (linoleic acid). Comparison of the activities of 9(Z)-octadecenoic acid and 12(Z)-octadecenoic acid implies that the two double bonds of linoleic acid contribute almost equally to the C-H bond-breaking step in the normal lipoxygenase reaction. The results are consistent with the notion that SBLO-1 functionalizes substrates by a radical mechanism.
Clapp
Charles H
CH
Department of Chemistry, Bucknell University, Lewisburg, Pennsylvania 17837, USA. cclapp@bucknell.edu
Strulson
Matthew
M
Rodriguez
Pamela C
PC
Lo
Richmond
R
Novak
Mark J
MJ
D000475:Alkenes / Q000378:metabolism; D000730:Androstadienes / Q000378:metabolism; D002384:Catalysis; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D008084:Lipoxygenase / Q000378:metabolism*; D019301:Oleic Acid / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity
D000475:Alkenes; D000730:Androstadienes; D005229:Fatty Acids, Monounsaturated; D019301:Oleic Acid; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D008084:Lipoxygenase; D005978:Glutathione; D010100:Oxygen