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.     

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.     

On the relationships of substrate orientation, hydrogen abstraction, and product stereochemistry in single and double dioxygenations by soybean lipoxygenase-1 and its Ala542Gly mutant

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-(3)H- and 11S-(3)H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.     

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 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).     

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.     

Stopped-flow kinetic investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the allosteric site

Herein, we report on the role of the allosteric site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The K(D) for the activation was determined to be 25.9 +/- 2.3 microM and the rate constant for the oxidation of the iron cofactor, k(2), to be 182 +/- 4 s(-1). Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a K(i) of 13.7 +/- 1.3 microM for the catalytic site and a K(i)' of 140 +/- 9 microM for the allosteric site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a K(i) of 17.5 +/- 3.8 microM. These results suggest that OS binds to an allosteric site that is separate from the catalytic iron site. We further observed that the allosteric site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS.     

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 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.     

Alkaline structural transition of 4-nitrobenz-2-oxa-1,3-diazolyl-Lysozyme. Kinetic and spectroscopic investigations.

When lysozyme is reacted with 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-CL), A 1:1 covalent product is produced, in which the NBD group arylates the phenolic hydroxyl group of Tyr-23 (Aboderin, A. A., and Boedefeld, E. (1976) Biochim. Biophys. Acta 420, 177). Changing the pH from neutral to alkaline conditions results in a large spectral shift of the absorption band associated with the NBD chromophore (Aboderin, A. A., Boedefeld, E., and Luisi, P. L. (1973) Biochim. Biophys. Acta 328, 30). In the present work it is shown that this spectral change is due to the formation of a sigma complex in which a hydroxyl ion is added to the aromatic nucleus of the nitrobenzoxadiazole system. Circular dichroic studies suggest that the NBD group is held in a conformationally rigid state in the protein. The kinetics of the spectral change accompanying the formation of the sigma complex has been investigated with a rapid mixing stopped flow spectrophotometer both in the modified enzyme and in the low molecular weight model compounds N-acetyl-(O-NBD)-L-tyrosinamide and glycyl-(O-NBD)-L-tyrosine. In the pH range from 10.1 to 12.7, the time course of the reaction is first order in the case of the modified enzyme (k = 4.8 s-1) and bimolecular and much slower (under pseudo-first order conditions) in the low molecular weight compounds. It is suggested that in the enzyme the reaction proceeds much faster because of the hydrophobic environment around the reacting groups. It is further suggested that the unimolecularity in the enzyme is due to a rate-determining isomerization step, probably connected with a local rearrangement of the protein conformation following the ionization of Tyr-20.     

Spectroscopic characterization of soybean lipoxygenase-1 mutants: the role of second coordination sphere residues in the regulation of enzyme activity

Lipoxygenases are non-heme iron enzymes, which catalyze the stereo- and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five- and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe(II) was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe(II) systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.     

Inhibition of soybean lipoxygenase-1 by n-alcohols and n-alkylthiols.

A series of n-alcohols and n-alkylthiols with carbon chains from 2 to 12 were examined for the inhibition of soybean lipoxygenase-1 (L-1). The alcohol produces a competitive inhibition, the extent of which increases with an increase in the carbon number of alkyl chain up to 8. Whereas the inhibition of the alkylthiol is noncompetitive, the extent of which is almost independent from the carbon number. From the behavior of pKi dependence on the carbon number of the alcohol, the decyl group appears to be optimum to bind to L-1. The thermodynamic analysis for the inhibition based upon van 't Hoff equation indicates positive enthalpy and entropy changes for the binding of the alcohol to the enzyme and negative enthalpy and positive to negative entropy changes for that of the alkylthiol. These observations suggest that the alcohol inhibits L-1 by binding of the hydrophobic alkyl tail to the catalytic site of the enzyme by a hydrophobic interaction. The alkylthiol inhibits by binding of the nucleophilic sulfhydryl head to a polarizable region of the enzyme and the alkyl tail to a hydrophobic region of the enzyme free from the steric hindrance as an anchor.     

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.     

Crystallization and preliminary x-ray analysis of soybean lipoxygenase-1, a non-heme iron-containing dioxygenase

Crystals of lipoxygenase-1 from soybeans have been grown by the method of vapor diffusion in the presence of sodium formate, ammonium acetate, and lithium chloride at pH 7.0. This enzyme contains a non-heme iron and is closely related to a human lipoxygenase found in leukocytes that participates in the biosynthesis of leukotrienes and lipoxins. The crystals are monoclinic space group C2 with cell dimensions of a = 183.8 A, b = 123.2 A, c = 94.3 A and beta = 102.9 degrees. They diffract beyond 2.7 A, are stable for several days in the x-ray beam, and appear to be suitable for x-ray diffraction studies.     

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.     

On the role of molecular oxygen in lipoxygenase activation: comparison and contrast of epidermal lipoxygenase-3 with soybean lipoxygenase-1

The oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) is associated with a lag phase during which the resting ferrous enzyme is converted to the active ferric form by reaction with fatty acid hydroperoxide. Epidermal lipoxygenase-3 (eLOX3) is atypical in displaying hydroperoxide isomerase activity with fatty acid hydroperoxides through cycling of the ferrous enzyme. Yet eLOX3 is capable of dioxygenase activity, albeit with a long lag phase and need for high concentrations of hydroperoxide activator. Here, we show that higher O(2) concentration shortens the lag phase in eLOX3, although it reduces the rate of hydroperoxide consumption, effects also associated with an A451G mutation known to affect the disposition of molecular oxygen in the LOX active site. These observations are consistent with a role of O(2) in interrupting hydroperoxide isomerase cycling. Activation of eLOX3, A451G eLOX3, and soybean LOX-1 with 13-hydroperoxy-linoleic acid forms oxygenated end products, which we identified as 9R- and 9S-hydroperoxy-12S,13S-trans-epoxyoctadec-10E-enoic acids. We deduce that activation partly depends on reaction of O(2) with the intermediate of hydroperoxide cleavage, the epoxyallylic radical, giving an epoxyallylic peroxyl radical that does not further react with Fe(III)-OH; instead, it dissociates and leaves the enzyme in the activated free ferric state. eLOX3 differs from soybean LOX-1 in more tightly binding the epoxyallylic radical and having limited access to O(2) within the active site, leading to a deficiency in activation and a dominant hydroperoxide isomerase activity.     

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.     

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.