(10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid methyl ester as an anti-inflammatory compound from Ehretia dicksonii

The methanol extract of Ehretia dicksonii provided (10E, 12Z, 15Z)-9-hydroxy-10,12,15-octadecatrienoic acid methyl ester (1) which was isolated as an anti-inflammatory compound. Compound 1 suppressed 12-Otetradecanoyl-phorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 microg (the inhibitory effect (IE) was 43%). Linolenic acid methyl ester did not inhibit this inflammation at the same dose. However, the related compounds of 1, (9Z,11E)-13hydroxy-9,11-octadecadienoic acid (5) and (9Z,llE)13-oxo-9,11-octadecadienoic acid (6), showed potent activity (IE500 microg of 63% and 79%, respectively). Compounds 1, 4 ((9Z, 12Z, 14E)-16-hydroxy-9,12,14-octadecatrienoic acid), 5 and 6 also showed inhibitory activity toward soybean lipoxygenase at a concentration of 10 microg/ml.     

A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1'Z)-12-(1'-hexenyloxy)-9,11-dodecadienoic [(omega5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of alpha-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1'Z,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dodecadienoic [(omega5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, (1)H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.     

Thermal conversions of trimethylsilyl peroxides of linoleic and linolenic acids

The trimethylsilyl (TMS) peroxides/esters of the fatty acid hydroperoxides (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPOD) and (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid (13-HPOT) were subjected to gas chromatography-mass spectrometry and products formed by thermal rearrangements were identified. The main products were decadienals and the TMS derivatives of 13-oxo-9,11-tridecadienoic acid, epoxyalcohols, hemiacetals, and ketodienes. Oxy radicals as well as epoxyallylic radicals served as intermediates in the formation of these compounds. The thermal TMS peroxide conversions documented provided biomimetic models for enzymatic conversions of fatty acid hydroperoxides and also offered a method to generate an array of oxylipin derivatives of value as reference compounds in GC-MS studies.     

Cyclization of natural allene oxide in aprotic solvent: formation of the novel oxylipin methyl cis-12-oxo-10-phytoenoate

Allene oxide, (9Z,11E)-12,13-epoxy-9,11-octadecadienoic acid (12,13-EOD), was prepared by incubation of linoleic acid (13S)-hydroperoxide with flaxseed allene oxide synthase (AOS) and purified (as methyl ester) by low temperature HPLC. Identification of pure 12,13-EOD was substantiated by its UV and (1)H NMR spectra and by GC-MS data for its methanol trapping product. The methyl ester of 12,13-EOD (but not the free carboxylic acid) is slowly cyclized in hexane solution, affording a novel cyclopentenone cis-12-oxo-10-phytoenoic acid. Free carboxylic form of 12,13-EOD does not cyclize due to the exceeding formation of macrolactone (9Z)-12-oxo-9-octadecen-11-olide. The spontaneous cyclization of pure natural allene oxide (12,13-EOD) into cis-cyclopentenone have been observed first time.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Allene oxide cyclase: a new enzyme in plant lipid metabolism

The mechanism of the biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid (12-oxo-PDA) from 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid in preparations of corn (Zea mays L.) was studied. In the initial reaction the hydroperoxide was converted into an unstable allene oxide, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, by action of a particle-bound hydroperoxide dehydrase. A new enzyme, allene oxide cyclase, catalyzed subsequent cyclization of allene oxide into 9(S),13(S)-12-oxo-PDA. In addition, because of its chemical instability, the allene oxide underwent competing nonenzymatic reactions such as hydrolysis into alpha- and gamma-ketol derivatives as well as spontaneous cyclization into racemic 12-oxo-PDA. (+/-)-cis-12,13-Epoxy-9(Z)-octadecenoic acid and (+/-)-cis-12,13-epoxy-9(Z),15(Z)-octadecadienoic acid, in which the epoxy group was located in the same position as in the allene oxide substrate, served as potent inhibitors of corn allene oxide cyclase. On the other hand, the isomeric (+/-)-cis-9,10-epoxy-12(Z)-octadecenoic acid had little inhibitory effect. Allene oxide cyclase was present in the soluble fraction of corn homogenate and had a molecular weight of about 45,000 as judged by gel filtration. The enzyme activity was detected in several plant tissues, the highest levels being observed in potato tubers and in leaves of spinach and white cabbage.     

Biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid: identification of an allene oxide cyclase

Incubation of 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid with corn (Zea mays L.) hydroperoxide dehydrase led to the formation of an unstable allene oxide derivative, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid. Further conversion of the allene oxide yielded two major products, i.e. alpha-ketol 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic acid, and 12-oxo-10,15(Z)-phytodienoic acid (12-oxo-PDA). 12-Oxo-PDA was formed from allene oxide by two different pathways, i.e. spontaneous chemical cyclization, leading to racemic 12-oxo-PDA, and enzyme-catalyzed cyclization, leading to optically pure 12-oxo-PDA. The allene oxide cyclase, a novel enzyme in the metabolism of oxygenated fatty acids, was partially characterized and found to be a soluble protein with an apparent molecular weight of about 45,000 that specifically catalyzed conversion of allene oxide into 9(S),13(S)-12-oxo-PDA.     

Human neutrophils convert the sebum-derived polyunsaturated fatty acid Sebaleic acid to a potent granulocyte chemoattractant

Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP(+), the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B(4), indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of its chemoattractant properties, sebum-derived 5-oxo-ODE could be involved in neutrophil infiltration in inflammatory skin diseases.     

Hydroperoxide-dependent epoxidation of unsaturated fatty acids in the broad bean (Vicia faba L.)

Incubation of linoleic acid with the 105,000g particle fraction of the homogenate of the broad bean (Vicia faba L.) led to the formation of the following products: 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid, 9,10-epoxy-12(Z)-octadecenoic acid (9(R),10(S)/9(S)/10(R), 80/20), 12,13-epoxy-9(Z)-octadecenoic acid (12(S),13(R)/12(R)/13(S), 64/36), and 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid (9(S),10(R)/9(R),10(S), 91/9). Oleic acid incubated with the enzyme preparation in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid or cumene hydroperoxide was converted into 9,10-epoxyoctadecanoic acid (9(R),10(S)/9(S),10(R), 79/21). Two enzyme activities were involved in the formation of the products, an omega 6-lipoxygenase and a hydroperoxide-dependent epoxygenase. The lipoxygenase, but not the epoxygenase, was inhibited by low concentrations of 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid. In contrast, the epoxygenase, but not the lipoxygenase, was readily inactivated in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid. Studies with 18O2-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid showed that the epoxide oxygens of 9,10-epoxyoctadecanoic acid and of 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid were derived from hydroperoxide and not from molecular oxygen.     

Soybean lipoxygenase-1 enzymically forms both (9S)- and (13S)-hydroperoxides from linoleic acid by a pH-dependent mechanism

Soybean lipoxygenase-1 produces a preponderance of two chiral products from linoleic acid, (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid and (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid. The former of these hydroperoxides was generated at all pH values, but in the presence of Tween 20, the latter product did not form at pH values above 8.5. As the pH decreased below 8.5, the proportion of (9S)-hydroperoxide increased linearly until at pH 6 it constituted about 25% of the chiral products attributed to enzymic action. Below pH 6, lipoxygenase activity was barely measurable, and the hydroperoxide product arose mainly from autoxidation and possibly non-enzymic oxygenation of the pentadienyl radical formed by the enzyme. The change in percent enzymically formed 9-hydroperoxide between pH 6.0 and 8.5 paralleled the pH plot of a sodium linoleate/linoleic acid titration. It was concluded that the (9S)-hydroperoxide is formed only from the nonionized carboxylic acid form of linoleic acid. Methyl esterification of linoleic acid blocked the formation of the (9S)-hydroperoxide by lipoxygenase-1, but not the (13S)-hydroperoxide. Since the hydroperoxydiene moieties of the (9S)- and (13S)-hydroperoxides are spatially identical when the molecules are arranged head to tail in opposite orientations, it is suggested that the carboxylic acid form of the substrate can arrange itself at the active site in either orientation, but the carboxylate anion can be positioned only in one orientation. These observations, as well as others in the literature, suggest and active-site model for soybean lipoxygenase-1.     

Detection of an enol intermediate in the hydroperoxide lyase chain cleavage reaction

Guava (Psidium guajava) hydroperoxide lyase (HPL) preparations were incubated with [1-(14)C](9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid for 1 min at 0 degrees C, followed by rapid extraction/trimethylsilylation. Analysis of the trimethylsilylated products by gas chromatography-mass spectrometry and radio-high-performance liquid chromatography revealed a single predominant (14)C-labelled compound, identified by its (1)H-nuclear magnetic resonance, ultraviolet and mass spectra as the trimethylsilyl ether/ester of (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid. Longer time incubations afford smaller yield of this enol due to its partial tautomerization into (9Z)-12-oxo-9-dodecenoic acid. The data obtained demonstrate that formation of (9Z)-12-oxo-9-dodecenoic acid in the HPL reaction is preceded by unstable enol oxylipin, and further suggest that hemiacetals are the true products of HPL catalysis.     

Occurrence of free and esterified lipoxygenase products in leaves of Glechoma hederacea L. and other Labiatae

Leaves of Glechoma hederacea L. and other Labiatae contain (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid, (10E,12Z,15Z)-9-oxo-10,12,15-octadecatrienoic acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid and (10E,12Z)-9-oxo-10,12-octadecadienoic acid in a ratio of 71/14/12/3 (by mass), predominantly esterified in the membrane ester lipids. The leaves contain the highest level of these products, whereas only small amounts were found in the stalk and the roots. The chemical structures of these compounds were established by ultraviolet and infrared spectroscopy, by co-chromatography with authentic standards on various types of HPLC columns including chiral-phase HPLC and gas chromatography/mass spectrometry. The stereochemical specificity indicates the enzymatic origin of the products, most probably via a lipoxygenase reaction. Freshly harvested specimens of G. hederacea L. contain only small amounts of hydroxy-polyenoic fatty acids. Air-drying causes a strong increase in the content of free and esterified (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid. Up to 80% of the hydroxy fatty acids of the total lipid extracts were esterified in the cellular lipids. The data presented indicate that lipoxygenase products occur in the cellular ester lipids of G. hederacea L. and other Labiatae. The results are discussed in the light of a possible involvement of the lipoxygenase pathway in the natural senescence of leaves.     

Identification of an allene oxide synthase (CYP74C) that leads to formation of alpha-ketols from 9-hydroperoxides of linoleic and linolenic acid in below-ground organs of potato

Allene oxide synthase (AOS) enzymes are members of the cytochrome P450 enzyme family, sub-family CYP74. Here we describe the isolation of three cDNAs encoding AOS from potato (StAOS1-3). Based on sequence comparisons, they represent members of either the CYP74A (StAOS1 and 2) or the CYP74C (StAOS3) sub-families. StAOS3 is distinguished from the other two AOS isoforms in potato by its high substrate specificity for 9-hydroperoxides of linoleic and linolenic acid, compared with 13-hydroperoxides, which are only poor substrates. The highest activity was shown with (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) as a substrate. This hydroperoxide was metabolized in vitro to alpha- and gamma-ketols as well as to the cyclopentenone compound 10-oxo-11-phytoenoic acid. They represent hydrolysis products of the initial StAOS3 product 9,10-epoxyoctadecadienoic acid, an unstable allene oxide. By RNA gel hybridization blot analysis, StAOS3 was shown to be expressed in sprouting eyes, stolons, tubers and roots, but not in leaves. StAOS3 protein was found in all organs tested, but mainly in stems, stolons, sprouting eyes and tubers. As in vivo reaction products, the alpha-ketols derived from 9-hydroperoxides of linoleic and linolenic acid were only found in roots, tubers and sprouting eyes. Immunolocalization showed that StAOS3 was associated with amyloplasts and leucoplasts.     

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa

The micro-alga Chlorella pyrenoidosa expresses an enzymatic activity that cleaves the 13-hydroperoxide derivatives of linoleic acid [13-hydroperoxy-9(Z),11(E)-octadecadienoic acid, 13-HPOD] and linolenic acid [13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid, 13-HPOT] into volatile C(5) and non-volatile C(13) oxo-products. This enzymic activity initially was attributed to a hydroperoxide lyase enzyme; however, subsequent studies showed that this cleavage activity is the result of lipoxygenase activity under anaerobic conditions. Headspace analysis of the volatile products by GC/MS showed the formation of pentane when the substrate was 13-HPOD, whereas a more complex mixture of hydrocarbons was formed when 13-HPOT was the substrate. Analysis of the non-volatile cleavage products from 13-HPOD by liquid chromatography/MS indicated the formation of 13-oxo-9(Z),11(E)-tridecadienoic acid (13-OTA) along with the 13-keto-octadecadienoic acid derivative. When the substrate is 13-HPOT, liquid chromatography/MS analysis indicated the formation of 13-OTA as the major non-volatile product. Aldehyde dehydrogenase (AldDH) oxidizes 13-OTA to an omega-dicarboxylic acid, whereas alcohol dehydrogenase (ADH) reduces 13-OTA to an omega-hydroxy carboxylic acid. AldDH and ADH require the oxidized (NAD(+)) and reduced (NADH) forms of the cofactor NAD, respectively. By combining the action of AldDH and ADH into a continuous cofactor-recycling process, it is possible to simultaneously convert 13-OTA to the corresponding omega-dicarboxylic acid and omega-hydroxy carboxylic acid derivatives.     

Characterization of a C-5,13-Cleaving Enzyme of 13(S)-Hydroperoxide of Linolenic Acid by Soybean Seed

An activity was found in mature soybean seeds (Glycine max L. cv Century) that cleaved 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13S-HPOT) into 13-oxo-9(Z),11(E)-tridecadienoic acid and two isomeric pentenols, 2(Z)-penten-1-ol and 1-penten-3-ol. Isomeric pentene dimers were also produced and were presumably derived from the combination of two pentene radicals. 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-HPOD) was, by contrast, a poor substrate. Activity with 13S-HPOT increased 24-fold under anaerobic conditions reminiscent of a similar anaerobic promoted reaction of 13S-HPOD catalyzed by lipoxygenase (LOX) in the presence of linoleic acid. However, prior to ion-exchange chromatography, cleavage activity did not require linoleic acid. After separation by gel filtration followed by ion-exchange chromatography, cleavage activity was lost but reappeared in the presence of either linoleic acid or dithiothreitol. Under these conditions cleavage activity was coincident with the activity of types 1 and 2 LOX. LOX inhibitors suppressed the cleavage reaction in a manner similar to inhibition of LOX activity. Heat-generated alkoxyl radicals derived from either 13S-HPOT or 13S-HPOD afforded similar products and yields of 13-oxo-9(Z),11(E)-tridecadienoic acid compared to the enzymic reaction. The product 1-penten-3-ol may be the precursor of the "raw-bean" volatile ethylvinylketone.     

Fatty acid allene oxides. II. Formation of two macrolactones from 12,13(S)-epoxy-9(Z),11-octadecadienoic acid

An unstable fatty acid allene oxide, 12,13(S)-epoxy-9(Z),11-octadecadienoic acid, was recently identified as the product formed from 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid in the presence of corn (Zea mays L.) hydroperoxide dehydrase (M. Hamberg (1987) Biochim. Biophys. Acta 920, 76-84). The present paper is concerned with the spontaneous decomposition of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid in acetonitrile solution. Two major products were isolated and characterized, i.e. macrolactones 12-keto-9(Z)-octadecen-11-olide and 12-keto-9(Z)-octadecen-13-olide.     

Hidden stereospecificity in the biosynthesis of divinyl ether fatty acids

Incubations of [8(R)-2H]9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid, [14(R)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid and [14(S)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid were performed with preparations of plant tissues containing divinyl ether synthases. In agreement with previous studies, generation of colneleic acid from the 8(R)-deuterated 9(S)-hydroperoxide was accompanied by loss of most of the deuterium label (retention, 8%), however, the opposite result (98% retention) was observed in the generation of 8(Z)-colneleic acid from the same hydroperoxide. Formation of etheroleic acid and 11(Z)-etheroleic acid from the 14(R)-deuterated 13(S)-hydroperoxide was accompanied by loss of most of the deuterium (retention, 7-8%), and, as expected, biosynthesis of these divinyl ethers from the corresponding 14(S)-deuterated hydroperoxide was accompanied by retention of deuterium (retention, 94-98%). Biosynthesis of omega5(Z)-etheroleic acid from the 14(R)- and 14(S)-deuterated 13(S)-hydroperoxides showed the opposite results, i.e. 98% retention and 4% retention, respectively. The experiments demonstrated that biosynthesis of divinyl ether fatty acids from linoleic acid 9- and 13-hydroperoxides takes place by a mechanism that involves stereospecific abstraction of one of the two hydrogen atoms alpha to the hydroperoxide carbon. Furthermore, a consistent relationship between the absolute configuration of the hydrogen atom eliminated (R or S) and the configuration of the introduced vinyl ether double bond (E or Z) emerged from these results. Thus, irrespective of which hydroperoxide regioisomer served as the substrate, divinyl ether synthases abstracting the pro-R hydrogen generated divinyl ethers having an E vinyl ether double bond, whereas enzymes abstracting the pro-S hydrogen produced divinyl ethers having a Z vinyl ether double bond.     

Identification of new geometric isomers of methyl linoleate hydroperoxide and their chromatographic behavior

New geometric isomers, methyl (9Z,11Z)-13-hydroperoxy-9,11-octadecadienoate and methyl (10Z,12Z)-9-hydroperoxy-10,12-octadecadienoate, were proved to be present in methyl linoleate hydroperoxide produced by autoxidation. They were identified from their UV, MS, and 1H-NMR spectra after conversion to the corresponding oxo derivatives: methyl (9Z,11Z)-13-oxo-9,11-octadecadienoate and methyl (10Z,12Z)-9-oxo-10,12-octadecadienoate. Their chromatographic behavior is described.     

Factors affecting the initial rate of lipoxygenase catalysis

The rate of peroxidation of linoleic acid by soybean type-1 lipoxygenase was studied under conditions which assured that the substrate was present as a monomolecular solution and that the first 5% of the reaction was observed. In order to achieve this, the kinetics were carried out at pH 10.0 in borate buffer using linoleic acid and enzyme concentrations of less than 75 μM and 0.2 nM respectively. The initial rate was increased by the presence of added product (13-hydroperoxy-9(Z),11(E)-octadecadienoic acid) in the substrate solutions in a concentration dependent and saturatable fashion. Product analogues lacking the hydroperoxide group (13-hydroxy-9(Z),11(E)-octadecadienoic acid and 13-methoxy-9(Z),11(E)-octadecadienoic acid) did not evoke this rate enhancing effect. These compounds reduced the initial rate when preincubated with enzyme prior to mixing with substrate. The results indicated that the chemical reactivity of the product was a necessary requirement for its activating effect on the enzyme.     

The "heterolytic hydroperoxide lyase" is an isomerase producing a short-lived fatty acid hemiacetal

To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10-60 s at 0 degrees C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1'-hydroxy-3'-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [(18)O(2)]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [(18)O(2)]13-HPOT retained two and one (18)O atoms, respectively, whereas no (18)O was incorporated from [(18)O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.