Soybean lipoxygenase-1 oxygenates synthetic polyenoic fatty acids with an altered positional specificity. Evidence for inverse substrate alignment

The positional specificity is the decisive enzyme property for classificati on of lipoxygenases and for the currently used lipoxygenase nomenclature. I t has been reported before that soybean lipoxygenase-1, which oxygenates po lyenoic fatty acids at alkaline pH to the corresponding n - 6 hydroperoxy d erivative, exhibits a different positional specificity when either the reac tion conditions or the substrate structure is altered. To investigate the i mpact of structural substrate modifications on the positional specificity o f this enzyme and to force an inverse substrate binding, we synthesized ara chidonic acid analogues modified at the co-terminus. Care was taken that th e double bond system remained unchanged so that hydrogen abstraction from a ll three bisallylic methylenes was theoretically possible. We found that co -modification of arachidonic acid leads to an impaired substrate affinity a nd a reduced reaction rate, but we did not detect any 5-lipoxygenation prod ucts, suggesting that structural modification of the co-end may not be suff icient to force an inverse substrate orientation. However, when both ends o f the fatty acid chain (a)terminus and free carboxylate) were modified simu ltaneously, a considerable share of 5-lipoxygenation products was detected. These results indicate that introduction of polar or bulky groups at the m ethyl terminus of polyenoic fatty acids was not sufficient to force an inve rse substrate orientation. However, simultaneous introduction of an omega - OH group and methylation of the carboxylate led to formation of significant 5-lipoxygenation products, suggesting an inverse head to tail substrate or ientation.