Alterations in Leukotriene Synthase Activity of the Human 5-Lipoxygenase by Site-Directed Mutagenesis Affecting Its Positional Specificity

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of lipoxygenases. Although the mechanistic basis of lipoxygenase specificity is not completely understood, sequence determinants for the positional specificity have been identified for various isoenzymes. In this study we altered the positional specificity of the human 5-lipoxygenase by multiple site-directed mutagenesis and assayed the leukotriene A4 synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A4 as indicated by the dominant formation of leukotriene A4 hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C10, it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A4 synthase activity. Surprisingly, we found that this quadruple mutant exhibited a similar leukotriene synthase activity as the wild-type enzyme in addition to its double oxygenation activity. The leukotriene synthase activity of the 8-lipoxygenating double mutant F359W + A424I was almost twice as high, and similar amounts of leukotriene A4 hydrolysis products and double oxygenation derivatives were detected with this enzyme species. These data indicate that site-directed mutagenesis of the human 5-lipoxygenase that leads to alterations in the positional specificity favoring arachidonic acid 15-lipoxygenation does not suppress the leukotriene synthase activity of the enzyme. The residual 8-lipoxygease activity of the mutant enzyme and its augmented rate of 5-HpETE conversion may be discussed as major reasons for this unexpected result.