| Abstract
| - For oxygenation of polyenoic fatty acids by 12- and 15-lipoxygenases the methyl terminus ofthe substrate constitutes the signal for the initial hydrogen abstraction. In contrast, for 5-lipoxygenasesan inverse head to tail substrate orientation has been proposed. However, recent structure-based sequencealignments suggested a conserved uniform substrate orientation for 5S- and 15S-lipoxygenation.Oxygenation of 15S-HETE derivatives by various wild-type and mutant lipoxygenases was investigated,and the evidence proved an inverse substrate orientation: (i) Substrate affinity and Vmax of 15S-HETEoxygenation by arachidonic acid 15-lipoxygenases are >1 order of magnitude lower than the correspondingdata for polyenoic fatty acids. 5S,15S- and 14R,15S-DiH(P)ETE were identified as major reaction products.(ii) Methylation of the carboxylate group of 15S-HETE augmented the reaction rate and shifted the reactionspecificity strongly toward 5S-lipoxygenation. In contrast, methyl arachidonate was less effectivelyoxygenated than the free acid. Methylation of 15S-HETrE(8,11,14), which lacks the C5−C6 doublebond, was without major impact on the oxygenation rate and on the product specificity. (iii) Introductionof a bulky glycerol moiety at the carboxylic group of 15S-HETE reversed the kinetic effects of methylationand led to a 14R-oxygenation of the substrate. (iv) When the product pattern of 15S-HETE oxygenationby the recombinant wild-type rabbit 15-lipoxygenase was compared with that formed by the Arg403Leumutant, 5S- and 8S-lipoxygenations were augmented and 14R,15S-DiH(P)ETE formation was impaired.(v) Phe353Leu or Ile418Ala mutation of the same enzyme, which favored 12S-HETE formation fromarachidonic acid, strongly augmented 8S-lipoxygenation of 15S-HETE methyl ester. These kinetic dataand the alterations in the product specificity are consistent with the concept of an inverse head to tailsubstrate orientation during the oxygenation of 15S-HETE methyl ester and/or of free 15S-HETE by15-LOXs. For 5S- and 8S-lipoxygenation, 15-HETE may slide into the substrate binding pocket with itscarboxy terminus approaching the doubly allylic methylenes C-7 or C-10 to the non-heme iron.
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