Investigation of the chemical conversion of hydroperoxyeicosatetraenoate to leukotriene epoxide using stereospecifically labeled arachidonic acid. Comparison with the enzymatic reaction

A series of stereospecifically labeled polyunsaturated fatty acids were prepared by biosynthesis from [8-DR-3H]- and [8-LS-3H]stearic acids. The labeled stearic acids were synthesized by a novel scheme employing readily available alkyne and aldehyde starting materials. The stereochemical purity of the prochiral tritium labels was judged to be greater than 99%, as determined by analysis of the octadec-1-yn-8(R)- and 8(S)-ol intermediates in the synthesis. Previously, the labeled arachidonic acids were used to investigate the stereoselectivity of hydrogen abstraction in the biosynthesis of leukotriene epoxides. We have now investigated the selectivity of hydrogen abstraction in a chemical synthesis of 14,15-leukotriene (LT) A4 from mixtures of [3-14C]- and either [10-DR-3H]- or [10-LS-3H]15(S)-HPETE methyl esters. Reaction with either chirally labeled precursor led to 70-95% retention of 3H relative to 14C in the 14,15-LTA4 and 10-Z-14,15-LTA4 products after purification by high performance liquid chromatography. The 15-dienone obtained from this reaction was consistently enriched in 3H relative to 14C after isolation and purification. Evidence was obtained to indicate that the majority of the 3H in the products was retained in its original location and configuration. These results indicate that the biomimetic chemical reaction is stereo-random with respect to hydrogen loss from carbon 10 and that, in contrast to the reaction as it occurs in leukocytes and platelets, in the chemical model the reaction begins by decomposition of the hydroperoxide group, with hydrogen loss from carbon 10 occurring as a late or final step.     

Specificity of an HPETE peroxidase from rat PMN

The 15,000xg supernatant of sonicated rat PMN contains 5-lipoxygenase that converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A4 and an HPETE peroxidase that catalyzes reduction of the 5-HPETE. The specificity of this HPETE peroxidase for peroxides, reducing agents, and inhibitors has been characterized to distinguish this enzyme from other peroxidase activities. In addition to 5-HPETE, the HPETE peroxidase will catalyze reduction of 15-hydroperoxyeicosatetraenoic acid, 13-hydroperoxyoctadecadienoic acid, and 15-hydroperoxy-8,11,13-eicosatrienoic acid, but not cumene or t-butylhydroperoxides. The HPETE peroxidase accepted 5 of 11 thiols tested as reducing agents. However, glutathione is greater than 15 times more effective than any other thiol tested. Other reducing agents, ascorbate, NADH, NADPH, phenol, p-cresol, and homovanillic acid, were not accepted by HPETE peroxidase. This enzyme is not inhibited by 10 mM KCN, 2 mM aspirin, 2 mM salicylic acid, or 0.5 mM indomethacin. When 5-[14C]HPETE is generated from [14C]arachidonic acid in the presence of unlabeled 5-HPETE and the HPETE peroxidase, the 5-[14C]HETE produced is of much lower specific activity than the [14C]arachidonic acid. This indicates that the 5-[14C]HPETE leaves the active site of 5-lipoxygenase and mixes with the unlabeled 5-HPETE in solution prior to reduction and is a kinetic demonstration that 5-lipoxygenase has no peroxidase activity. Specificity for peroxides, reducing agents, and inhibitors differentiates HPETE peroxidase from glutathione peroxidase, phospholipid-hydroperoxide glutathione peroxidase, a 12-HPETE peroxidase, and heme peroxidases. The HPETE peroxidase could be a glutathione S-transferase selective for fatty acid hydroperoxides.     

Specificity of an HPETE peroxidase from rat PMN

The 15,000xg supernatant of sonicated rat PMN contains 5-lipoxygenase that converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A₄ and an HPETE peroxidase that catalyzes reduction of the 5-HPETE. The specificity of this HPETE peroxidase for peroxides, reducing agents, and inhibitors has been characterized to distinguish this enzyme from other peroxidase activities. In addition to 5-HPETE, the HPETE peroxidase will catalyze reduction of 15-hydroperoxyeicosatetraenoic acid, 13-hydroperoxyoctadecadienoic acid, and 15-hydroperoxy-8,11,13-eicosatrienoic acid, but not cumene or t-butylhydroperoxides. The HPETE peroxidase accepted 5 of 11 thiols tested as reducing agents. However, glutathione is >15 times more effective than any other thiol tested. Other reducing agents, ascorbate, NADH, NADPH, phenol, p-cresol, and homovanillic acid, were not accepted by HPETE peroxidase. This enzyme is not inhibited by 10 mM KCN, 2 mM aspirin, 2 mM salicylic acid, or 0.5 mM indomethacin. When 5-[¹⁴C]HPETE is generated from [¹⁴C]arachidonic acid in the presence of unlabeled 5-HPETE and the HPETE peroxidase, the 5-[¹⁴C]HETE produced is of much lower specific activity than the [¹⁴C]arachidonic acid. This indicates that the 5-[¹⁴C]HPETE leaves the active site of 5-lipoxygenase and mixes with the unlabeled 5-HPETE in solution prior to reduction and is a kinetic demonstration that 5-lipoxygenase has no peroxidase activity. Specificity for peroxides, reducing agents, and inhibitors differentiates HPETE peroxidase from glutathione peroxidase, phospholipid-hydroperoxide glutathione peroxidase, a 12-HPETE peroxidase, and heme peroxidases. The HPETE peroxidase could be a glutathione S-transferase selective for fatty acid hydroperoxides.     

Specific high affinity binding of lipoxygenase metabolites of arachidonic acid by liver fatty acid binding protein

Liver fatty acid binding protein (L-FABP) binds avidly the arachidonic acid metabolites, hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs). Binding of 15-[3H]HPETE was specific, saturable, reversible, and rapid. Protein specificity was indicated by the following order: L-FABP greater than bovine serum albumin greater than ovalbumin = beta-lactoglobulin greater than ribonuclease. Ligand specificity was evidenced by the following order of apparent competition: 15-HPETE greater than or equal to 5-HETE greater than or equal to 5-HPETE = oleic acid greater than 12-HETE greater than 12-HPETE greater than or equal to 15-HETE greater than prostaglandin E1 much greater than leukotriene C4 greater than prostaglandin E2 much greater than thromboxane B2 = leukotriene B4. Once bound, 15-HPETE was reversibly displaced. Ligand was recovered from the protein complex and confirmed to be 15-[3H]HPETE by TLC. L-FABP bound HPETE with a dissociation constant of 76 nM,5-HETE at 175 nM, and 15-HETE at 1.8 microM, and the reference fatty acids oleic acid at 1.2 microM and arachidonic acid at 1.7 microM. Thus, the affinity was approximately 16-fold greater for 15-HPETE, and 7-fold higher for 5-HETE, than for oleic acid. The need exists for studies of complexes of L-FABP with the HPETEs and HETEs in hepatocytes, especially since L-FABP has previously been associated with mitosis in normal hepatocytes, and shown to be the target protein of two liver carcinogens, and these arachidonic acid metabolites have been found to be able to modulate activities related to cell growth.     

Formation of 14,15-hepoxilins of the A(3) and B(3) series through a 15-lipoxygenase and hydroperoxide isomerase present in garlic roots.

We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the "A"-type and the "B"-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S), 15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5-10-fold less active than 11(S), 12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.     

A secondary isotope effect in the lipoxygenase reaction

Fatty acids containing a prochiral tritium label have often been used in the study of enzymatic reactions which involve an obligatory step of hydrogen abstraction. In the lipoxygenase reaction, the primary isotope effect associated with this approach is detected as an isotopic enrichment of the substrate. Herein we characterize a previously unrecognized secondary isotope effect which changes the specific activity of both the substrate and product. The 12-lipoxygenase of human platelets removes the 10-LS hydrogen of arachidonic acid in the formation of 12-hydroperoxyeicosatetraenoic acid. We studied the specific activity changes associated with conversion of the enantiomerically labeled [10-DR-3H]arachidonic acid to 12-[10-3H]hydroxyeicosatetraenoic acid in aspirin-treated platelets. [3-14C]Arachidonic acid served as internal standard. The most pronounced change in 3H/14C ratio in the early stages of reaction was a 15-20% deficiency of tritium in the product. Later, the remaining arachidonate showed a marked increase in 3H/14C ratio. The changes in specific activity closely matched those predicted for a secondary isotope effect. Comparison of these data with the theoretical equations for a secondary isotope effect indicated the 10-DR-3H substrate reacted at about 84% of the rate of unlabeled molecules. Interestingly, this secondary isotope effect is similar in magnitude to the secondary isotope effect in autoxidation reactions, a finding compatible with a basic similarity in reaction mechanisms in enzymatic and non-enzymatic oxygenation of lipids.     

Hydroxylation of specifically deuterated limonene enantiomers by cytochrome p450 limonene-6-hydroxylase reveals the mechanism of multiple product formation

The regiochemistry and facial stereochemistry of the limonene-6-hydroxylase- (CYP71D18-) mediated hydroxylation of the monoterpene olefin limonene are determined by the absolute configuration of the substrate. (-)-(4S)-Limonene is hydroxylated at the C6 allylic position to give (-)-trans-carveol as the only product, whereas (+)-(4R)-limonene yields multiple hydroxylation products with (+)-cis-carveol predominating. Specifically deuterated limonene enantiomers were prepared to investigate the net stereospecificity of hydroxylation at C6 and the mechanism of multiple product formation. The results of isotopically sensitive branching experiments of competitive and noncompetitive design were consistent with a nondissociative kinetic mechanism, indicating that (4R)-limonene has sufficient freedom of motion within the active site of CYP71D18 to allow formation of either the trans-3- or cis-6-hydroxylated product. However, the kinetic isotope effects resulting from deuterium abstraction were significantly smaller than expected for an allylic hydroxylation, and they did not approach the intrinsic isotope effect. (4S)-Limonene is oxygenated with almost complete stereospecificity for hydrogen abstraction from the trans-6-position, demonstrating rigid orientation during hydrogen abstraction and hydroxyl delivery. The oxygenation of (4R)-limonene leading to the formation of (+/-)-trans-carveol is accompanied by considerable allylic rearrangement and stereochemical scrambling, whereas the formation of (+)-cis-carveol proceeds without allylic rearrangement and with nearly complete stereospecificity for hydrogen abstraction from the cis-6-position. These results demonstrate that a single cytochrome P450 enzyme catalyzes the hydroxylation of small antipodal substrates with distinct stereochemistries and reveal that substrate-dependent positional motion of the intermediate carbon radical (and, therefore, hydroxylation stereospecificity) is determined by active-site binding complementarity. Thus, epimerization and allylic rearrangement are not inherent features of these reactions but occur when loss of active-site complementarity allows increased substrate mobility.     

Mechanistic studies of the dioxygenase and leukotriene synthase activities of the porcine leukocyte 12S-lipoxygenase

Lipoxygenases react with hydroperoxy fatty acids and catalyze dioxygenase or dehydrase (leukotriene A4 (LTA4) synthase) types of reactions. In the present investigation we studied the mechanism of reaction of the purified porcine leukocyte 12S-lipoxygenase with 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE). Oxygen-18 labeling experiments with GC-MS analysis were used to distinguish dioxygenase and leukotriene synthase activities of the enzyme; 8S,15S-DiHPETE and 14R,15S-DiHPETE were formed by oxygenation, and a series of 8,15- and 14,15-diols were formed via enzymatic synthesis of 14,15-LTA4 and nonenzymatic hydrolysis of the epoxide. 10D-3H- and 10L-3H-labeled substrates were used to study the stereospecificity of the C-10 hydrogen abstraction in the synthesis of these products. Formation of 14,15-DiHPETE and 14,15-LTA4 was associated with stereoselective abstraction of hydrogen from the 10L position of 15S-HPETE. The same type of measurements on the 8S,15S-DiHPETE product indicated a variable (50-250%) retention of the 10L-3H label, and a consistent 90% retention of the 10D-3H. In contrast, the synthesis of 8S,15S-DiHPETE by the soybean lipoxygenase was associated with the expected stereoselective abstraction of the 10D hydrogen. It appears that the porcine leukocyte 12S-lipoxygenase synthesizes 8S,15S-DiHPETE by a different mechanism.     

Stereochemical trends in copper amine oxidase reactions

Copper amine oxidases (EC 1.4.3.6) exhibit atypical stereochemical patterns in the reactions they catalyze. Dopamine and tyramine are oxidized with abstraction of the pro-R hydrogen by the porcine plasma amine oxidase, the pro-S hydrogen by pea seedling amine oxidase and a net nonstereospecific proton abstraction by the bovine plasma enzyme. This provides the first example in which a reaction catalyzed by enzymes in the same formal class occurs by all three possible stereochemical routes. To assess the underlying mechanistic significance of this heterogeneity, we have established the stereochemical course of the oxidation of tyramine by five additional copper amine oxidases using 1H NMR spectroscopy. Reactions catalyzed by rabbit and sheep serum amine oxidases are nonstereospecific. These enzymes exhibit rare mirror image binding with differential flux through two opposite and stereospecific reaction pathways. Differential primary kinetic isotope effects are observed for each mode, 8 and 4.6 for pro-S abstraction and 2.6 and 2.7 for pro-R abstraction by the sheep and rabbit amine oxidases, respectively. Tyramine oxidations catalyzed by the soybean and chick pea amine oxidases and porcine kidney diamine oxidase, however, are all stereospecific, occurring with loss of the pro-S hydrogen at C-1. Solvent exchange profiles are consistent within each stereochemical class of enzyme; the pro-R and nonstereospecific enzymes exchange solvent into C-2 of product aldehydes, the pro-S enzymes do not.     

Stereospecific elimination of hydrogen at C-10 in eicosapentaenoic acid during the conversion to leukotriene C5

(10L)- and (10D)-[1-14C, 10-3H]5,8,11,14,17-eicosapentaenoic acids were synthesized to investigate mechanistic and stereochemical aspects of leukotriene biosynthesis. Experiments with mastocytoma cells showed that a hydrogen is stereospecifically eliminated from C-10 during the conversion of eicosapentaenoic acid to leukotriene C5. The hydrogen lost has the pro-S (D) configuration. 5-Hydroxy-6,8,11,14,17-eicosapentaenoic acid, formed in the same experiments, was enriched in tritium when the (10D), but not when the (10L), isomer of labeled eicosapentaenoic acid was used. This indicates that oxygenation of the acid at C-5 occurred before the elimination of hydrogen and suggests that removal of the pro-S hydrogen at C-10 in 5-hydroperoxy-6,8,11,14,17-eicosapentaenoic acid initiates its transformation to trans-5(S),6(S)-oxido-7,9-trans-11,14,17-cis-eicosapentaenoic acid (leukotriene A5).     

Selective quantification of arachidonic acid hydroperoxides and their hydroxy derivatives in reverse-phase high performance liquid chromatography

For the quantification of lipid hydroperoxides by high performance liquid chromatography (HPLC), it has been necessary to improve the detection system specific to the hydroperoxy group. We first developed a technique which combined detection by uv absorption due to conjugated diene and detection based on electrochemical (EC) reduction in reverse-phase HPLC for the selective determination of arachidonic acid hydroperoxides (hydroperoxyeicosatetraenoic acid, HPETE) and its reduced derivative, hydroxyeicosatetraenoic acid (HETE). 15-HPETE was quantified selectively by EC detection, although both 15-HPETE and 15-HETE were detected by uv absorption and were hardly resolved in the chromatogram. Isomers in HPETE obtained from autoxidized arachidonic acid were partially separated in the chromatogram and seem to have been quantified similarly to 15-HPETE. The application of this analytical system to the analysis of 15-HPETE added in human plasma has demonstrated that the recovery of HPETE extracted from human plasma is much lower than that from normal saline and that HPETE is reduced to HETE by incubation at 37 degrees C. The fact that a high concentration of glutathione accelerated this reduction may indicate that human plasma possesses a glutathione-dependent HPETE-reducing ability as a defense system against excess accumulation of lipid hydroperoxides. Blood plasma effectively suppressed the decomposition of HPETE induced by ferrous ion indicating the presence of factors which prevent the action of ferrous ion on HPETE.     

The autoxidation of glyceraldehyde and other simple monosaccharides under physiological conditions catalysed by buffer ions

Glyceraldehyde and other simple monosaccharides autoxidise under physiological conditions generating 1-hydroxyalkyl (carbon-centred) free radicals and intermediates of dioxygen reduction: superoxide, hydrogen peroxide and hydroxyl radicals. The major glyceraldehyde-derived product is the alpha-ketoaldehyde, hydroxypyruvaldehyde. Close similarities between the temperature dependence of the kinetics of glyceraldehyde autoxidation and glyceraldehyde enolisation to an ene-diol indicates that enolisation is the rate-determining step in the autoxidative process. Inspection of a wide range of carbonyl compounds showed that the monosaccharide moiety -CH(OH)-C- is conserved in carbonyl compounds reactive towards autoxidation, indicating that the ability to form an ene-diol is a prerequisite to monosaccharide autoxidation. The ene-diol intermediate autoxidises rapidly to the products: hydrogen peroxide, water and alpha-ketoaldehydes: beta-hydroxypyruvaldehyde is produced from glyceraldehyde and dihydroxyacetone, glyoxal from glycolaldehyde autoxidation. Ene-diol autoxidation is catalysed by hydrogen peroxide and trace metal ion contaminants; removal of either of these factors sufficiently retards ene-diol autoxidation such that ene-diol autoxidation rather than enolisation becomes the rate determining step in the overall autoxidative process. Under enolisation control, the rate of monosaccharide autoxidation is influenced by pH and the buffer system used for pH control.     

Novel biological transformations of 15-Ls-hydroperoxy-5,8,11,13-eicosatetraenoic acid

[1-14C]Arachidonic acid was incubated with homogenates of the fungus, Saprolegnia parasitica. The products consisted of comparable amounts of two epoxy alcohols, 15-Ls-hydroxy-11,12-epoxy-5cis,8cis,13trans- eicosatrienoic acid and 15-hydroxy-13,14-epoxy-5cis,8cis,11cis-eicosatrienoic acid. Results of incubations carried out in the presence of nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, p-hydroxymercuribenzoate as well as glutathione peroxidase plus reduced glutathione demonstrated that transformation of arachidonic acid into epoxy alcohols occurred with the formation of 15-Ls-hydroperoxy-5cis,8cis,11cis,13trans- eicosatetraenoic acid (15-HPETE) as an intermediate. The pathway involved a lipoxygenase catalyzing the oxygenation of arachidonic acid at the 15L position to produce 15-HPETE, and a hydroperoxide isomerase activity which catalyzed conversion of 15-HPETE into the two epoxy alcohols. Studies with 15-[18O2]HPETE demonstrated that both oxygens of 15-HPETE were retained in the epoxy alcohols. Furthermore, experiments with mixtures of 15-[18O2]-and 15-[16O2]HPETE showed that conversion of 15-HPETE into epoxy alcohols occurred by an intramolecular transfer of hydroperoxide oxygen.     

Hydroperoxy-arachidonic acid mediated n-hexanal and (Z)-3- and (E)-2-nonenal formation in Laminaria angustata

In higher plants, C6 and C9 aldehydes are formed from C18 fatty acids, such as linoleic or linolenic acid, through formation of 13- and 9-hydroperoxides, followed by their stereospecific cleavage by fatty acid hydroperoxide lyases (HPL). Some marine algae can also form C6 and C9 aldehydes, but their precise biosynthetic pathway has not been elucidated fully. In this study, we show that Laminaria angustata, a brown alga, formed C6 and C9 aldehydes enzymatically. The alga forms C9 aldehydes exclusively from the C20 fatty acid, arachidonic acid, while C6 aldehydes are derived either from C18 or from C20 fatty acid. The intermediates in the biosynthetic pathway were trapped by using a glutathione/glutathione peroxidase system, and subjected to structural analyses. Formation of (S)-12-, and (S)-15-hydroperoxy arachidonic acids [12(S)HPETE and 15(S)HPETE] from arachidonic acid was confirmed by chiral HPLC analyses. These account respectively for C9 aldehyde and C6 aldehyde formation, respectively. The HPL that catalyzes formation of C9 aldehydes from 12(S)HPETE seems highly specific for hydroperoxides of C20 fatty acids.     

Stereochemical course of phosphokinases. The use of adenosine [gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase.

We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.     

Kinetics of leukotriene A4 synthesis by 5-lipoxygenase from rat polymorphonuclear leukocytes

When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.     

Leukotriene formation by a purified reticulocyte lipoxygenase enzyme. Conversion of arachidonic acid and 15-hydroperoxyeicosatetraenoic acid to 14, 15-leukotriene A4

The purified lipoxygenase of rabbit reticulocytes converts arachidonic acid at 0 degrees C to 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) via reactions which involve hydrogen abstraction at C-13 and C-10, respectively. At 37 degrees C the enzyme converts arachidonic acid to additional products which were identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, 8,15-dihydroperoxy-5,9,11,13- and 5,15-dihydroperoxy-6, 6,8,11,13-eicosatetraenoic acids (8,15-diHPETE and 5,15-HPETE, respectively) and diastereoisomers of 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-diHPETEs). The 8,15- and 5,15-diHPETEs were formed by double lipoxygenation since each incorporated 2 molecules of 18O2 and since their synthesis from 15-HPETE was blocked under anaerobic conditions. The 8,15-diHETEs each incorporated 18O from 18O2 at C-15 and were found to arise from nonenzymatic hydrolysis of an epoxytriene which was identified as 14,15-leukotriene A4 by trapping in acidic methanol. This compound was a major product of 15-HPETE in anaerobic incubations. The conversion of 15-HPETE to 14,15-leukotriene A4 was inhibited by the lipoxygenase inhibitors nordihydroguairetic acid and 5,8,11,14-eicosatetraynoic acid. The 14,15-leukotriene A4 synthase and 15-lipoxygenase activities were inhibited by 5,8,11,14-eicosatetraynoic acid in a similar time-dependent manner. The results support a mechanism whereby 14,15-leukotriene A4 is synthesized from 15-HPETE by a further enzymatic step carried out by the reticulocyte 15-lipoxygenase via hydrogen abstraction at C-10 and a redox cycle of the non-heme iron atom of the enzyme.     

12-Lipoxygenase from bovine polymorphonuclear leukocytes, an enzyme with leukotriene A4-synthase activity

Bovine polymorphonuclear leukocytes exhibit a 12-lipoxygenase activity upon sonication. In contrast to bovine platelet 12-lipoxygenase and other 12-lipoxygenases, this enzyme is unable to convert 5(S)-HETE (5(S)-hydroxy,6-trans-8,11,14-cis-eicosatetraenoic acid) or 5(S)-HPETE (5(S)-hydroperoxy,6-trans-8,11,14-cis-eicosatetraenoic acid) into 5(S),12(S)-dihydroxy-6,10-trans,8,14-cis-eicosatetraenoic acid. Surprisingly, the formation of leukotriene A4-derived products namely leukotriene B4 and the leukotriene B4-isomers 12-epi,6-trans- leukotriene B4 and 6-trans-leukotriene B4, was observed upon incubation of this enzyme with 5(S)-HPETE. Hence, the 12-lipoxygenase from bovine polymorphonuclear leukocytes possesses leukotriene A4-synthase activity.     

Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases

Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30-80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2.     

5-Lipoxygenase and leukotriene synthesis: effects of calcium ions and of inhibitors

5-Lipoxygenase and leukotriene (LT) A4 synthase, the first two enzymes in the pathway converting arachidonic acid to leukotrienes, can be co-purified. The Ca2+-activated conversion of arachidonic acid and of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to LTB4 have been compared, using cytosol from human leucocytes. The two activities showed identical responses to a number of inhibitors, suggesting that the two catalytic activities may involve the same active centre. The effects of Ca2+ ions were further investigated. With 5-HPETE as substrate, substantial synthesis of LTB4 was given in the absence of Ca2+, and the inhibitor sensitivity of this component was quite different from that of the Ca2+-activated component. This Ca2+-independent synthase activity was, however, very low in saponin-permeabilised washed leucocytes and it may therefore be not significant physiologically. With arachidonic acid as substrate at pH 7, the activity was highly Ca2+-dependent at a low substrate concentration (6.6 microM), but at a high concentration (132 microM) substantial activity was observed without Ca2+. This was also found when 5-lipoxygenase was assayed in cytosol from RBL cells. At pH 8-8.5, however, Ca2+ was required at both high and low concentrations of arachidonic acid. This suggests that Ca2+ is required for 5-lipoxygenase activity on arachidonate ions in solution but possibly not on protonated arachidonic acid or micelles.