Cationic substrates of soybean lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate. The manner in which SBLO-1 binds substrates is uncertain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion (LTMA) to give primarily13(S)-hydroperoxy-9(Z),11(E)-octadecadienyltrimethylammonium ion. The rate of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate substrates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates.     

Irreversible inactivation of soybean lipoxygenase-1 by hydrophobic thiols

Soybean lipoxygenase-1 is inactivated by micromolar concentrations of the following hydrophobic thiols: 1-octanethiol, 12(S)-mercapto-9(Z)-octadecenoic acid (S-12-HSODE), 12(R)-mercapto-9(Z)-octadecenoic acid (R-12-HSODE), and 12-mercaptooctadecanoic acid (12-HSODA). In each case, inactivation is time-dependent and not reversed by dilution or dialysis. Inactivation requires 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13-HPOD), which suggests that it is specific for the ferric form of the enzyme. Lipoxygenase catalyzes an oxygenation reaction on each of the aforementioned thiols, as judged by the consumption of O(2). These reactions also require 13-HPOD. 1-Octanethiol is converted to 1-octanesulfonic acid, which was identified by GC/MS of its methyl ester. The rates of oxygen uptake for R- and S-12-HODE are about 5- and 2.5-fold higher than the rate with 1-octanethiol. The stoichiometries of inactivation imply that inactivation occurs on approximately 1 in 18 turnovers for 12-HSODA, 1 in 48 turnovers for 1-octanethiol, 1 in 63 turnovers for S-12-HSODE, and 1 in 240 turnovers for R-12-HSODE. These data imply that close resemblance to lipoxygenase substrates is not a crucial requirement for either oxidation or inactivation. Under the conditions of our experiments, inactivation was not observed with several more polar thiols: mercaptoethanol, dithiothreitol, L-cysteine, glutathione, N-acetylcysteamine, and captopril. The results imply that hydrophobic thiols irreversibly inactivate soybean lipoxygenase by a mechanism that involves oxidation at sulfur.     

Inhibition of soybean lipoxygenase-1 by 10-butyryl substituted 1,8-dihydroxy-9-anthrone (butantrone)

10-Butyryl substituted 1,8-dihydroxyanthrone (butantrone) inhibited soybean lipoxygenase-1 irreversibly and more efficiently than its parent compound 1,8-dihydroxyanthrone (dithranol, anthralin) (IC50 values 0.090 mM and 1.1 mM, respectively). Intact butantrone rather than its hydrolysis product was the primary effector and the 10-butyryl moiety its site specific probe, probably directing the inhibitor to the proximity of the binding site of the lipid substrate/product.     

The interaction of nitric oxide with soybean lipoxygenase-1.

The interaction of nitric oxide with the non-heme iron dioxygenase lipoxygenase is reported. This apparently resulted in a novel type of complex where an electron is donated to the NO molecule. In addition a new position for an EPR transition from iron was discovered which, it is suggested results from high spin ferric iron in a field of axial symmetry characterised by a very low value for D.     

Effect of endocannabinoids on soybean lipoxygenase-1 activity

Endocannabinoids appear to be involved in a variety of physiological processes. Lipoxygenase activity has been known to be affected by unsaturated fatty acids or phenolic compounds. In this study, we examined whether endocannabinoids containing both N-acyl group and phenolic group can affect the activity of soybean lipoxygenase (LOX)-1, similar to mammalian 15-lipoxygenase in physicochemical properties. First, N-arachidonoyl dopamine and N-oleoyl dopamine were found to inhibit soybean LOX-1-catalyzed oxygenation of linoleic acid in a non-competitive manner with a K_i value of 3.7 μM and 6.2 μM, respectively. Meanwhile, other endocannabinoids failed to show a remarkable inhibition of soybean LOX-1. Separately, N-arachidonoyl dopamine and N-arachidonoyl serotonin were observed to inactivate soybean LOX-1 with K_in value of 27 μM and 24 μM, respectively, and k₃ value of 0.12 min⁻¹ and 0.35 min⁻¹, respectively. Furthermore, such an inactivation was enhanced by ascorbic acid, but suppressed by 13(S)-hydroperoxy-9,11-octadecadienoic acid. Taken together, it is proposed that endocannabinoids containing polyunsaturated acyl moiety and phenolic group may be efficient for the inhibition as well as inactivation of 15-lipoxygenase.     

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.     

Inhibition of endotoxin-induced monocytic procoagulant activity by n-alcohols and anesthetics.

1. The effect of n-alcohols (methanol and ethanol) and anesthetics (lidocaine, thiopental, methohexital and thiamylal) on procoagulant activity (PCA) in human peripheral-blood monocytes and non-adherent cultured leukemia promonocytic U937 and THP-1 cells was examined herein. 2. Exposure of whole blood to ethanol showed no effect on PCA in human monocytes. However, ethanol dose-dependently inhibited LPS-induced PCA in isolated human monocytes. 3. In THP-1 cells, ethanol had no significant effect on PCA in either non-challenged or LPS-induced status. However, the induction of PCA by LPS was substantially inhibited when cells were pretreated with 1% ethanol (v/v) for 72 hr. 4. In U937 cells, n-alcohols and anesthetics resulted in dose-dependent depressions in PCA. Importantly, the percent reduction in LPS-induced PCA was much more pronounced than that in non-challenged PCA. 5. These data clearly suggest that n-alcohols and anesthetics readily inhibit the LPS-stimulatory action on monocytic PCA.     

Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1.

1. The EPR spectrum at 15 degrees K of soybean lipoxygenase-1 in borate buffer pH 9.0 has been studied in relation to the presence of substrate (linoleic acid), product (13-L-hydroperoxylinoleic acid) and oxygen. 2. The addition of 13-L-hydroperoxylinoleic acid to lipoxygenase-1 at pH 9.0 gives rise to the appearance of EPR lines at g equals 7.5, 6.2, 5.9 and 2.0, and an increased signal at g equals 4.3. 3. In view of the effect of the end product on both the kinetic lag period of the aerobic reaction and the fluorescence of the enzyme, it is concluded that 13-L-hydroperoxylinoleic acid is required for the activation of soybean lipoxygenase-1. Thus it is proposed that the enzyme with iron in the ferric state is the active species. 4. A reaction scheme is presented in which the enzyme alternatingly exists in the ferric and ferrous states for both the aerobic and anaerobic reaction.     

Conversion of 9-D- and 13-L-hydroperoxylinoleic acids by soybean lipoxygenase-1 under anaerobic conditions.

Soybean lipoxygenase-1 reacts with both 9-D and 13-L-hydroperoxylinoleic acids under anaerobic conditions. Approximately 40% of the hydroperoxide is converted into oxodienes, absorbing at 285 nm. Concomitantly, more polar compounds are formed, tentatively identified as being mainly epoxy-hydroxy-octadecenoic acids. When oxygen is present, the reaction is strongly inhibited, until in a very slow reaction the oxygen has been depleted. This accounts for the occurrence of a lag period.     

Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.

Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln(495) and Gln(697), which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn(694)). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln(495). The nature of the C-H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH(-) is the active site base. A new transition state model for hydrogen atom abstraction is proposed.     

Isolation of soybean lipoxygenase-2 by affinity chromatography

Isoenzyme lipoxygenase-2 from soybean was isolated by affinity chromatography. Gel electrophoresis showed it to be a single protein. Its pH optimum of 6.5, range of 5.0–8.0 and activity which increased when Ca²⁺ was added identified the isolated enzyme as lipoxygenase-2.     

Effect of denaturants on the structural properties of soybean lipoxygenase-1.

The effect of chemical (urea) and physical (temperature and high pressure) denaturation on the structural properties of soybean lipoxygenase-1 (LOX1) was analyzed through dynamic fluorescence spectroscopy and circular dichroism. We show that the fluorescence decay of the native protein could be fitted by two lorentzian distributions of lifetimes, centered at 1 and 4 ns. The analysis of the urea-denatured protein suggested that the shorter distribution is mostly due to the tryptophan residues located in the N-terminal domain of LOX1. We also show that a pressure of 2400 bar and a temperature of 55 degrees C brought LOX-1 to a state similar to a recently described stable intermediate "I." Analysis of circular dichroism spectra indicated a substantial decrease of alpha-helix compared with beta-structure under denaturing conditions, suggesting a higher stability of the N-terminal compared with the C-terminal domain in the denaturation process.     

Kinetic studies of oxygen reactivity in soybean lipoxygenase-1

The reactivity of O(2) with soybean lipoxygenase-1 (SLO) has been examined using a range of kinetic probes. We are able to rule out diffusional encounter of O(2) with protein, an outer-sphere electron transfer to O(2), and proton transfer as rate-limiting steps in k(cat)/K(M)(O(2)) for wild-type enzyme (WT SLO); this restricts the rate-limiting step to either the combination of O(2) with L(*) or a subsequent conformational change. In the Ile(553) --> Phe mutant, which constricts the putative O(2) binding channel [Knapp et al. (2001) J. Am. Chem. Soc. 123, 2931-2932], k(cat)/K(M)(O(2)) decreases by over a factor of 20; yet, this mutant appears to have the same rate-limiting step as WT SLO. It is argued that the slow step on k(cat)/K(M)(O(2)) is the combination of O(2) with L(*), with proximal protein effects determining the rate of reaction. The available data for SLO support the view that enzymes can affect O(2) reactivity without a direct involvement of metal cofactors. The primary role of the Fe(3+) cofactor is to generate an enzyme-bound radical, while the protein is concluded to control the stereo- and regiochemistry of O(2) encounter with this radical.     

Kinetic isotope effects in the oxidation of arachidonic acid by soybean lipoxygenase-1

The reaction of soybean lipoxygenase-1 with linoleic acid has been extensively studied and displays very large kinetic isotope effects. In this work, substrate and solvent kinetic isotope effects as well as the viscosity dependence of the oxidation of arachidonic acid were investigated. The hydrogen atom abstraction step was rate-determining at all temperatures, but was partially masked by a viscosity-dependent step at ambient temperatures. The observed KIEs on k(cat) were large ( approximately 100 at 25 degrees C).     

The effects of soybean lipoxygenase-1 on chloroplasts from wheat

Chloroplasts were isolated from primary leaves of wheat 12 days after germination and incubated at 25° for 45 min in the dark with soybean lipoxygenase-1. The lipoxygenase action was evident from a weak oxygen uptake of ca 0.18, μmol/hr per mg chloroplast protein. The lipoxygenase treatment caused a marked decrease in the photochemical activity, as measured by the reduction rate of 2,6-dichlorophenolindophenol. However, both the content and composition of the lipids as well as those of total fatty acids remained largely unchanged except for a slight but significant decrease in the total linolenic acid content. It is proposed that soybean lipoxygenase-1 selectively attacks free linolenic acid present in chloroplasts, followed by a chlorophyll-catalysed reaction of hydroperoxylinolenic acid with components of the electron transfer system.     

The effect of modification of sulfhydryl groups in soybean lipoxygenase-1

Soybean lipoxygenase-1 was found to contain five free sulfhydryl groups and no disulfide bridges. Three sulfhydryl groups react readily with methylmercuric halides. This modification results in significant changes of the catalytic properties of the enzyme. Comparison of modified and native lipoxygenase-1 shows the following: 1. The catalytic constant of the oxygenation of linoleic acid is reduced by approximately 50%, whereas the affinity towards linoleic acid remains unaltered. 2. At high concentrations of substrate and low concentrations of enzyme the kinetic lag phase in the oxygenation is considerably longer. 3. The regio- and stereospecificities of the oxygenation are significantly lower. 4. Besides hydroperoxides, oxo-octadecadienoic acids (4%) are formed during the oxygenation. 5. The cooxidation capacity is considerably enhanced. Treatment of methylmercury-modified lipoxygenase-1 with NaHS results in the complete recovery of the sulfhydryl groups and of the catalytic properties.     

Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1

Recently, it has been shown that lipoxygenase (LO) products affect the substrate specificity of human 15-LO. In the current paper, we demonstrate that soybean LO-1 (sLO-1) is not affected by its own products, however, inhibitors which bind the allosteric site, oleyl sulfate (OS) and palmitoleyl sulfate (PS), not only lower catalytic activity, but also change the substrate specificity, by increasing the arachidonic acid (AA)/linoleic acid (LA) ratio to 4.8 and 4.0, respectively. The fact that LO inhibitors can lower activity and also change the LO product ratio is a new concept in lipoxygenase inhibition, where the goal is to not only reduce the catalytic activity but also alter substrate selectivity towards a physiologically beneficial product.