Lipoxygenases catalyse the oxidation of polyunsaturated fatty acids and have been invoked in many diseases including cancer, atherosclerosis and Alzheimer's disease. Currently, no X-ray structures are available with substrate or substrate analogues bound in a productive conformation. Such structures would be very useful for examining interactions between substrate and active site residues. Reported here are the syntheses of linoleic acid analogues containing a sulfur atom at the 11 or 14 positions. The key steps in the syntheses were the incorporation of sulfur using nucleophilic attack of metallated alkynes on electrophilic sulfur compounds and the subsequent stereospecific tantalum-mediated reduction of the alkynylsulfide to the cis-alkenylsulfide. Kinetic assays performed with soybean lipoxygenase-1 showed that both 11-thialinoleic acid and 14-thialinoleic acid were competitive inhibitors with respect to linoleic acid with K(i) values of 22 and 35 microM, respectively. On the other hand, 11-thialinoleic acid was a noncompetitive inhibitor with respect to arachidonic acid with K(is) and K(ii) values of 48 and 36 microM, respectively. 11-Thialinoleic acid was also a noncompetitive inhibitor of human 15-lipoxygenase-1 with arachidonic acid (K(is) = 11.4 microM, K(ii) = 18.1 microM) or linoleic acid as substrate (K(is) = 20.1 microM, K(ii) = 20.0 microM), and a competitive inhibitor of human 12-lipoxygenase with arachidonic acid as substrate (K(i) = 2.5 microM). The presence of inhibitor did not change the regioselectivity of soybean lipoxygenase-1, human 12- or 15-lipoxygenase-1.

Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30-80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2.