Irreversible inhibition of soybean lipoxygenase-1 by hydroperoxy acids as substrates

Soybean lipoxygenase-1 was irreversibly inactivated by various peroxy acids containing a cis,cis-1,4-pentadiene group. Among these compounds, 15(S)-hydroperoxyeicosatetraenoic acid (15(S)-HPETE)2 was found to be the most effective in the inactivation of lipoxygenase. Although the prior exposure of 15(S)-HPETE to hemoglobin abolished the inhibitory effect of 15(S)-HPETE, the simultaneous inclusion of hemoglobin potentiated the inactivation of lipoxygenase by 15(S)-HPETE alone. Interestingly, the potentiating effect of hemoglobin was observed only in the incubations with peroxy acids possessing the cis,cis-1,4-pentadiene. In either the presence or the absence of hemoglobin, it was commonly observed that the enzyme inactivation, which was maximal at pH 10, was significantly protected by tocopherol, but neither by mannitol nor ethanol, and that the inclusion of arachidonic acid or linoleic acid prevented the enzyme inactivation. Based on these results, it is suggested that the selective inactivation of lipoxygenase by these peroxy acids may be due to unstable intermediates produced from hydroperoxy acids bound to the active site of lipoxygenase.     

Ketones as electrophilic substrates of lipoxygenase

The rate-limiting step of the lipoxygenase reaction involves the abstraction of a hydrogen from the methylene carbon of a 1,4-diene. One possibility for the mechanism of the enzyme is the abstraction of this hydrogen as a proton to generate a carbanionic intermediate or transition state. In order to investigate this possibility, 5-, 8-, 12-, and 15-hydroxy-eicosatetraenoic acid were oxidized to the corresponding ketones and these ketones were assayed as substrates of the 5-, 12-, and 15-lipoxygenases from rat neutrophils, rat platelets, and soybeans, respectively. The ketones were in no case better substrates than arachidonic acid and in some cases the hydroxyeicosatetraenoic acids were equally active as the corresponding ketones. Since no increased rate of oxidation for these electrophilic substrates was observed, it is concluded that no transition state with carbanionic character is generated in the rate-determining step of the lipoxygenase reaction.     

Keto fatty acids not containing doubly allylic methylenes are lipoxygenase substrates

The soybean lipoxygenase I oxygenates the unusual substrate 12-keto-(9Z)-octadecenoic acid methyl ester as indicated by oxygen uptake and spectral changes of the incubation mixture. The main oxygenation products have been isolated by HPLC and identified as 9,12-diketo-(10E)-octadecenoic acid methyl ester and 12-keto-(10E)-dodecenoic acid methyl ester by UV and IR spectroscopy, cochromatography with an authentic standard, gas chromatography/mass spectroscopy, and 1H NMR. In the formation of both compounds the oxygenase and hydroperoxidase activities of the enzyme appear to be involved. These data and the earlier results on the oxygenation of furanoic fatty acids (Boyer et al., 1979) indicate that the lipoxygenase reaction is not restricted to substrates containing a 1,4-pentadiene structure.     

Substrate specificity of potato lipoxygenase

The substrate specificity of potato lipoxygenase was examined using a partially purified enzyme preparation from tubers of a potato variety with low lipolytic acyl hydrolase activity. Potato lipoxygenase is fully active only on free linoleic acid or linolenic acid, and only acts directly on more complex glyceride moieties in the absence of any significant endogenous lipolytic acyl hydrolase activity.     

Secondary alkyl hydroperoxides as inhibitors and alternate substrates for lipoxygenase

Lipoxygenase plays a central role in polyunsaturated fatty acid metabolism, inaugurating the biosynthesis of eicosanoids in animals and phytooxylipins in plants. Redox cycling of the non-heme iron cofactor represents a critical element of the catalytic mechanism. Paradoxically, the isolated enzyme contains Fe(II), but the catalytically active form contains Fe(III), and the natural oxidant for the iron is the hydroperoxide product of the catalyzed reaction. Controlling the redox state of lipoxygenase iron with small molecules, inhibitors or activators, could be a means to modulate the activity of the enzyme. The effects of secondary alkyl hydroperoxides and the corresponding alcohols on soybean lipoxygenase-1 reaction rates were investigated and found to be very different. Secondary alcohols were noncompetitive or linear mixed inhibitors with inhibition constants in the millimolar concentration range, with more hydrophobic compounds producing lower values. Secondary alkyl hydroperoxides were inhibitors of lipoxygenase-1 primarily at high substrate concentration. They were more effective inhibitors than the alcohols, with dissociation constants in the micromolar concentration range. The hydroperoxides bearing longer alkyl substituents were the more effective inhibitors. Oxidation of the iron in lipoxygenase-1 by 2-hydroperoxyalkanes was evident in electron paramagnetic resonance (EPR) measurements, but the enzyme was neither activated nor was it inactivated. Instead there was evidence for an entirely different reaction catalyzed by the enzyme, a homolytic dehydration of the hydroperoxide to produce the corresponding carbonyl compound.     

Inactivation of soybean lipoxygenase 1 by 12-iodo-cis-9-octadecenoic acid

12-Iodo-cis-9-octadecenoic acid (12-IODE) is a time-dependent, irreversible inactivator of soybean lipoxygenase 1. The rate of inactivation is independent of 12-IODE concentration above 20 microM and is half-maximal at about 4 microM. Inactivation by 12-IODE requires lipid hydroperoxide, which must be present even after the initial oxidation of the iron in the enzyme from ferrous to ferric. Inactivation by 12-IODE is also dependent on O2. These findings suggest that 12-IODE is converted by the enzyme into a more reactive species, which is responsible for inactivation. No inactivation has been detected with 12-iodooctadecanoic acid, 12-bromo-cis-9-octadecenoic acid, 12-iodo-trans-9-octadecenoic acid, or a mixture of stereoisomers of 9,11-octadecadienoic acid.     

Kinetics of suicide substrates

In the light of the scheme presented by Walsh et al. (1978, Meth. Enzymol. 53, 437-448), Waley (1980, Biochem. J. 185, 771-773) and Tatsunami et al. (1981, Biochim. biophys. Acta, 662, 226-235) made a detailed study for the kinetics of suicide substrates. However, the effects of the enzyme reaction intermediate and product were not considered in their studies. Equations for the kinetics of suicide substrate were derived for a more reasonable scheme. A plotting method was proposed to determine the kinetic parameters and it has been shown that suicide substrate can be used to study the formative mechanism of enzyme-catalyzed reaction intermediate.     

Mode of utilization of amino acids as growth substrates by Azospirillum brasilense

The study was undertaken to analyze the rate of uptake and utilization of various amino acids by Azospirillum brasilense Sp81 (RG) in a basal mineral salts solution under non-nitrogen fixing condition. These amino acids including other nitrogenous compounds were tested for both N- and C-sources. The kinetic constants (Km and Vmax) of uptake of some amino acids (e.g. lysine, arginine, proline, glutamine and glutamic acid) were exploited using a Hanes-Woolf plot, and discussed in the context of nitrogen starvation or both carbon and nitrogen starvation. To summarize all the kinetic data for these amino acids strongly suggested that the mode of these amino acids utilization in this bacterium followed the same general pattern, although the quantitative differences were there. A single amino acid was able to satisfy the nitrogen needs of this bacterium in basal mineral salts solution, and this possibility could be considered for the cost-effective growth medium for this bacterium in the biotechnological industry.     

Enantiomeric separation and analysis of unsaturated hydroperoxy fatty acids by chiral column chromatography-mass spectrometry

Hydroperoxides of 18:2n-6 and 20:4n-6 were obtained by autoxidation and photooxidation. The enantiomers were separated as free acids (Reprosil Chiral-NR column, eluted with hexane containing 1-1.2% alcoholic modifier) and analyzed by on line UV detection (234nm) and liquid chromatography-MS/MS/MS of carboxylate anions (A(-)-->(A(-)-18)-->full scan) in an ion trap. The combination of UV and MS/MS/MS analysis facilitated identification of hydroperoxides even in complex mixtures of autoxidized or photooxidized fatty acids. The signal intensities increased about two orders of magnitude by raising the isolation width of A(-) from 1.5amu to 5 or 10amu for cis-trans conjugated hydroperoxy fatty acids, and one order of magnitude or more for non-conjugated hydroperoxy fatty acids. The S enantiomer of 8-, 9-, 10-, and 13-hydroperoxyoctadecadienoic acids and the S enantiomer of cis-trans conjugated hydroperoxyeicosatetraenoic acids eluted before the corresponding R enantiomer with two exceptions (11-hydroperoxylinoleic acid and 8-hydroperoxyeicosa-5Z,9E,11Z,14Z-tetraenoic acid). The separation of enantiomers or regioisomers could be improved by the choice of either isopropanol or methanol as alcoholic modifier.     

Kinetics of suicide substrates.

When suicide substrates inactivate enzymes during catalysis, formation of product and inactivation of enzyme proceed concurrently. The steady-state hypothesis is applicable when catalytic quantities of enzyme are used. Equations for the rate of inactivation have been derived and integrated to obtain equations describing progress curves.     

Transient-phase kinetics of enzyme inactivation induced by suicide substrates

This paper deals with the kinetic study of reaction mechanisms with enzyme inactivation induced by a suicide substrate in the presence or absence of an auxiliary substrate and in conditions of excess of substrates in relation to the enzyme concentration and vice versa. A transient-phase approach has been developed that enables explicit equations with one or two significant exponentials to be obtained, thereby showing the dependence of product concentration on time. The validity of these equations has been checked, and a comparison made with those previously obtained by other authors. We propose an experimental design to determine the corresponding parameters and kinetic constants. The simplicity of our method allows a systematic application to more complex mechanisms.     

Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.     

Regulation of tobacco lipoxygenase by methyl jasmonate and fatty acids

Lipoxygenase (LOX) activity and gene expression have been described previously to be induced in tobacco by fungal infection and elicitor treatment. We now report that LOX activity is induced in tobacco cell suspensions by treatment with methyl jasmonate (MeJa). This compound had no effect on the in vitro activity of tobacco LOX. Induction of LOX activity is a dose-dependent response with a maximum around 890 μM MeJa. Linolenic acid, the precursor for jasmonate synthesis, also induces LOX activity. When applied together with fungal elicitor, linolenic acid drastically increases and prolongs the induction of LOX activity. LOX activity and gene expression in elicited tobacco cells are partially inhibited by pretreatment with eicosatetraynoic acid (ETYA), a potent inhibitor of tobacco LOX in vitro. The induction by methyl jasmonate, in contrast, was not inhibited by ETYA pretreatment. These data suggest that induction of LOX gene expression and activity upon elicitation are regulated at least partially by LOX products. © Académie des Sciences/ Elsevier, Paris     

Inactivation of Ascaris suum by short-chain fatty acids

Ascaris suum eggs were inactivated in distilled water and digested sludge by butanoic, pentanoic, and hexanoic acids. The fatty acids (short-chain fatty acids [SCFA]) were effective only when protonated and at sufficient concentrations. The conjugate bases were not effective at the concentrations evaluated. Predictions from an inhibition model (50% inhibitory concentration [IC(50)]) based on quantitative structure-activity relationships were congruent with inactivation data.