title: Alterations in leukotriene synthase activity of the human 5-lipoxygenase by site-directed mutagenesis affecting its positional specificity.
abstract: 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 A(4) synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicos atetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A(4) as indicated by the dominant formation of leukotriene A(4) hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C(10), it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A(4) 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 A(4) 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.
authors.affiliation: Institute of Biochemistry, University Clinics Charite, Humboldt University, Hessische Strasse 3-4, 10115 Berlin, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Schwarz
authors.forename: C
authors.initials: C
authors.lastname: Gerth
authors.forename: M
authors.initials: M
authors.lastname: Anton
authors.forename: H
authors.initials: H
authors.lastname: Kuhn
mesh_terms: D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D017572:Leukotriene A4 / Q000378:metabolism*; D015289:Leukotrienes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D017572:Leukotriene A4; D015289:Leukotrienes; D011994:Recombinant Proteins; C025348:arachidonic acid 5-hydroperoxide; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase