Positional specificity of a Lupinus albus lipoxygenase in relation to enzyme concentration and effect of a double dioxygenation product of arachidonic acid.

When arachidonic acid was incubated with Lupinus Albus lipoxygenase, a trienoic fatty acid, 8,15-dihydroperoxy-5,9,11,13-eicosatetraenoic acid was formed in addition to the three HPETEs, 15-HPETE, 8-HPETE and 5-HPETE. The formation of the two major monohydroperoxy acids (15- and 5-HPETE) has been shown to depend on enzyme concentration. At low enzyme concentration (less than or equal to 1 unit), Lupinus Albus lipoxygenase displays its highest regiospecificity at pH 5.8, resulting in the formation of 5-HPETE as the major product (80%). As the enzyme concentration increased, the proportion of 5-HPETE decreased, while the one of 15-HPETE increased. On the other hand, the preincubation of the enzyme with 8,15-diHPETE led to the gradual inactivation of the lipoxygenase activity. The inhibitory effect of 8,15-diHPETE was abolished by using high enzyme concentration. Based on these observations, it is proposed that the trienoic fatty acid, 8,15-diHPETE stemmed from further lipoxygenation of 15-HPETE, blocked the formation of the 15(S) hydroperoxide by Lupinus Albus lipoxygenase but not the formation of 5(S) hydroperoxide.