Oxidation of linoleyl alcohol by potato tuber lipoxygenase: kinetics and positional, stereo, and geometrical (cis, trans) specificity of the reaction

The dioxygenation of linoleyl alcohol (LAL) by potato tuber lipoxygenase leads to formation of two positional isomeric products--9- and 13-hydroperoxyoctadecadien-1-ols (Butovich, I. A., Luk'yanova, S. M., and Reddy, C. C. (1998) Biochem. Biophys. Res. Commun. 249, 344-349). In the present study, we examined the stereospecificity and double-bond conformation of primary dioxygenation products of LAL catalyzed by potato lipoxygenase. In contrast to the product profiles of linoleic acid oxidation by potato lipoxygenase, oxidation of LAL led to all possible positional (9- and 13-), stereo, and geometrical (cis,trans and all-trans) isomers in equimolar mixtures at 25 degrees C. The reaction appears to proceed through an enzyme-catalyzed formation of a pentadiene carbon-centered radical followed by resonance stabilization of the radical and molecular oxygen insertion in an enzyme-dependent as well as an enzyme-independent pathway. A strict positional, stereo, and geometrical specificity of the dioxygenation products of LAL oxidation appears to be maintained when the reaction occurs at the active site of the enzyme. However, when the pentadiene carbon-centered radical of LAL is dissociated from the active site of the enzyme, it appears to be nonenzymatically transformed into a mixture of all possible positional and geometrical stereoisomers of primary dioxygenation products. The latter pathway was effectively blocked by the free radical scavenger 4-hydroxy-TEMPO, which substantially reduced the production of all-trans hydroperoxyoctadecadienols. In the presence of the scavenger, 9(S)-hydroperoxy-10E,12Z-octadecadien-1-ol was the predominant LAL oxidation product, representing approximately 80% of the total conjugated dienes, with 13(S)-hydroxy-9Z,11E-octadecadien-1-ol the expected product of reverse orientation of the substrate at the active site, accounting for approximately 10%. A similar pattern in oxidation of LAL was observed when the reactions were carried out at 0 degrees C.     

11-hydroperoxyeicosatetraenoic acid is the major dioxygenation product of lipoxygenase isolated from hairy root cultures of Solanum tuberosum.

The profile of primary dioxygenation products of arachidonic acid catalyzed by lipoxygenase isolated from hairy root cultures of Solanum tuberosum treated with a fungal elicitor was compared to that obtained for the enzyme from potato tubers. 11-Hydroperoxyeicosatetraenoic acid (11-HPETE) was the most abundant dioxygenation product formed followed by 8- and 5-HPETEs in the decreasing order of abundance. In contrast, 5-HPETE is the predominant oxidation product of lipoxygenase from potato tubers. Differences in the defense requirements of storage tuber as compared to roots may be the basis of the differences in regio-specificity demonstrated in this work.     

Properties of a Lipoxygenase in Green Algae (Oscillatoria sp.)

A lipoxygenase preparation was obtained from green algae Oscillatoria sp. and was shown to differ from previous described lipoxygenases in the positional specificity and pH characteristics of the dioxygenation reaction. The enzyme had a pH optimum at 8.8 and was inactive at pH 6. Oscillatoria lipoxygenase converted linoleic acid into two products: 13-hydroperoxylinoleic acid (52%) and 9-hydroperoxylinoleic acid (48%). The molecular weight of the enzyme was estimated at 124,000. Esculetin was found to be the best inhibitor of the enzyme activity.     

Kinetic study of lipoxygenase-hydroperoxylinoleic acid interaction.

Interaction of lipoxygenase with hydroperoxylinoleic acid, which is the product of this enzyme reaction and acts as an activator, was studied kinetically by the fluorescence stopped-flow method. The kinetic features are consistent with a two-step mechanism involving a fast bimolecular association process followed by a slow unimolecular process. The dissociation constant of the bimolecular process was 3 (+/-2) - 10(-5) M, which was appreciably dependent on temperature and pH, in contrast to the rate constant of the latter process. The enthalpy and the entropy of activation for the unimolecular process were estimated to be 21 kcal/mol and 20 e.u., respectively. The pH dependence of the rate constant indicated that an ionizable group with pK of about 8.6 is involved in the interaction. Linoleic acid, the substrate of lipoxygenase, and oleic acid inhibited the interaction between the lipoxygenase and the hydroperoxylinoleic acid by reducing the rate. A series of saturated monohydric alcohols also reduced the rate of the interaction as the chain length of the alcohols increases, though methanol and ethanol increased the rate of the interaction.     

Potato 5-lipoxygenase. Kinetics of linoleic acid oxidation

The role of main factors influencing the rate of potato 5-lipoxygenase oxidation of linoleic acid was investigated. It was found that nonionic detergent lubrol PX inhibited the potato lipoxygenase. Optimal pH for the linoleic acid oxidation was 6.3 temperature--45 degrees C and substrate concentration--3 x 10(-4) M (if lubrol PX was 0.02%). It was shown that potato 5-lipoxygenase was allosteric enzyme which possessed positive cooperativity for linoleic acid. The Hill coefficient was calculated (n = 1.40 +/- 0.15) with S0.5 = 75 +/- 10 microM.     

Double hydroperoxidation of α-linolenic acid by potato tuber lipoxygenase

The potato tuber lipoxygenase preparations convert α-linolenic acid not only to 9(S)-HPOTE, but also to some more polar metabolites. Two of these polar products, I and II, with ultraviolet absorbance maxima at 267 nm were purified by HPLC. It was found that metabolites I and II have, respectively, one and two hydroperoxy groups. Products of NaBH₄ reduction of both I and II were identified by their chemical ionization and electron impact mass spectra and by ¹H-NMR spectra as 9,16-dihydroxy-10(E), 12(Z), 14(E)-octadecatrienoic acid. The obtained results suggest that compound II is 9,16-dihydroperoxy-10(E), 12(Z), 14(E)-octadecatrienoic acid and product I is a mixture of two positional isomers, 9-hydroxy-16-hydroperoxy-10(E),12(Z),14(E)-octadecatrienoic and 9-hydroperoxy-16-hydroxy-10(E),12(Z), 14(E)-octadecatrienoic acids. Lipoxygenase converts efficiently [¹⁴C]9-HOTE into product I. Also, both metabolites I and II are the products of double dioxygenation. The second oxygenation at C-16 position as well as the first one at C-9 is controlled by lipoxygenase.     

A one-step method of 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid synthesis by soybean lipoxygenase

A product of lipoxygenase (LOX) oxidation of docosahexaenoic acid (DHA), 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diH(P)DHA] was obtained through various reaction pathways that involved DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z,13Z,15E,19Z-enoic acid [17(S)-H(P)DHA], soybean lipoxygenase (sLOX), and potato tuber lipoxygenase (ptLOX) in various combinations. The structure of the product was confirmed by HPLC, ultraviolet (UV) light spectrometry, GC-MS, tandem MS, and NMR spectroscopy. It has been found that 10,17(S)-diH(P)DHA formed by sLOX through direct oxidation of either DHA or 17(S)-H(P)DHA was apparently identical to the product of ptLOX oxidation of the latter. The sLOX- and ptLOX-derived samples of 10,17-diHDHAs coeluted under the conditions of normal, reverse, and chiral phase HPLC analyses, displayed identical UV absorption spectra with maxima at 260, 270, and 280 nm, and had similar one-dimensional and two-dimensional proton NMR spectra. Analysis of their NMR spectra led to the conclusion that 10,17-diHDHA formed by sLOX had solely 11E,13Z,15E configuration of the conjugated triene fragment, which was identical to the previously published structure of its ptLOX-derived counterpart. Based on the cis,trans geometry of the reaction products, the conclusion is made that in the tested conditions sLOX catalyzed direct double dioxygenation of DHA. Compared with the previously described two-enzyme method that involved sLOX and ptLOX, the current simplified one-enzyme procedure uses only sLOX as the catalyst of both dioxygenation steps.     

Interaction between 5-lipoxygenase and allosteric effector--sodium dodecyl sulfate

The role of allosteric effector--sodium dodecyl sulfate (SDS) in the lipoxygenase catalysis in micelle system has been studied. The effect of the stable hydrophobic bis-nitroxides, blocking the free radical transformation, on the oxidation of linoleic acid or linoleic alcohol by 5-lipoxygenase from potato tuber has been investigated. The inhibiting effect of nitroxide compounds on oxidation of linoleic acid or linoleic alcohol by 5-lipoxygenase depends on SDS concentration. The inhibition percentage is determined by the substrate nature and presence of allosteric effector. The presence of SDS did not lead to an appreciable change in the pKa values of ionogenic enzyme groups. The effect of SDS and micellar system on thermodynamic parameters for thermoinactivation of 5-lipoxygenase was studied. It was found that thermoinactivation rate constants and activation energy of enzyme thermoinactivation were increased in the presence of SDS. It is suggested that interaction of 5-lipoxygenase and allosteric effector--SDS intensifies the dissociation of radical intermediates from the active site of the enzyme. These findings are of physiological significance in the light of the lipoxygenase involvement in the membrane lipid peroxidation.     

Enzyme-catalyzed and enzyme-triggered pathways in dioxygenation of 1-monolinoleoyl-rac-glycerol by potato tuber lipoxygenase

It was shown for the first time that potato tuber lipoxygenase (ptLOX) catalyzed the aerobic oxidation of 1-monolinoleoyl-rac-glycerol (mLG) in a mixed micellar reaction solution with the non-ionic detergent monododecyl ether of decaoxyethylene glycol. No hydrolysis of mLG occurred during the reaction. The four major reaction products obtained at 23 degrees C were identified as 1-[9-hydroperoxy-10E,12Z-octadecadienoyl]-rac-glycerol (9-(E,Z)HPODE-GE, 41%), 1-[13-hydroperoxy-9Z,11E-octadecadienoyl]-rac-glycerol (13-(Z,E)-HPODE-GE, 17%), and their all-trans isomers ( approximately 21% each). The molar fraction of all-trans isomers depended on the temperature of the reaction solution; it was found that at 0 degrees C their molar fractions were approximately 15.5% each, while 9-(E,Z)HPODE-GE and 13-(Z,E)-HPODE-GE gave 42% and 27%, respectively, of the overall product. A free radical scavenger, 4-hydroxy-TEMPO, dramatically increased the molar fraction of 9-(E,Z)HPODE-GE, yielding 83% at 23 degrees C, at the expense of all other products. Chiral HPLC of 9-(E,Z)HPODE-GE formed in the presence of 4-hydroxy-TEMPO revealed that it was composed of approximately 94% S and approximately 6% (R) isomers. This assures largely a uniform orientation of mLG molecules in the ptLOX active center, with their methyl end most likely deepened into the protein globule. The second major product, 13-(Z,E)-HPODE-GE, which yielded approximately 9% of the total product formed in the presence of 4-hydroxy-TEMPO, was racemic, and so were the all-trans isomers. Therefore, the last three cannot be considered the true products of the enzyme reaction, which is known to be stereospecific. It appears that they were formed as a result of (i) leakage of the pentadienyl radicals from the ptLOX active center and their subsequent non-enzymatic dioxygenation, and/or (ii) leakage of the peroxyl radicals leading to a free radical chain reaction affording all positional, geometrical and stereoisomers of the products. This reaction resembles ptLOX oxidation of another non-ionizable substrate, linoleyl alcohol [I.A. Butovich, S.M. Luk'yanova, C.C. Reddy, Arch. Biochem. Biophys. 378 (2000) 65-77], and differed substantially from oxidation of ionizable linoleic acid. Consequently, formation of large amounts of the non-specific oxidation products might be considered a universal characteristic of ptLOX oxidation of non-ionizable compounds.     

Purification and Characterization of 3-Methylcrotonyl-Coenzyme A Carboxylase from Higher Plant Mitochondria

3-Methylcrotonyl-coenzyme A (CoA) carboxylase was purified to homogeneity from pea (Pisum sativum L.) leaf and potato (Solanum tuberosum L.) tuber mitochondria. The native enzyme has an apparent molecular weight of 530,000 in pea leaf and 500,000 in potato tuber as measured by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate disclosed two nonidentical subunits. The larger subunit (B subunit) is biotinylated and has an apparent molecular weight of 76,000 in pea leaf and 74,000 in potato tuber. The smaller subunit (A subunit) is biotin free and has an apparent molecular weight of 54,000 in pea leaf and 53,000 in potato tuber. The biotin content of the enzyme is 1 mol/133,000 g of protein and 1 mol/128,000 g of protein in pea leaf and potato tuber, respectively. These values are consistent with an A4B4 tetrameric structure for the native enzyme. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8 to 8.3 and at 35 to 38[deg]C in the presence of Mg2+. Kinetic constants (apparent Km values) for the enzyme substrates 3-methylcrotonyl-CoA, ATP, and HCO3- were: 0.1 mM, 0.1 mM, and 0.9 mM, respectively, for pea leaf 3-methylcrotonyl-CoA carboxylase and 0.1 mM, 0.07 mM, and 0.34 mM, respectively, for potato tuber 3-methylcrotonyl-CoA carboxylase. A steady-state kinetic analysis of the carboxylase-catalyzed carboxylation of 3-methylcrotonyl-CoA gave rise to parallel line patterns in double reciprocal plots of initial velocity with the substrate pairs 3-methylcrotonyl-CoA plus ATP and 3-methylcrotonyl-CoA plus HCO3- and an intersecting line pattern with the substrate pair HCO3- plus ATP. It was concluded that the kinetic mechanism involves a double displacement. Purified 3-methylcrotonyl-CoA carboxylase was inhibited by end products of the reaction catalyzed, namely ADP and orthophosphate, and by 3-hydroxy-3-methylglutaryl-CoA. Finally, as for the 3-methylcrotonyl-CoA carboxylases from mammalian and bacterial sources, plant 3-methylcrotonyl-CoA carboxylase was sensitive to sulfhydryl and arginyl reagents.     

N-dealkylation of aminopyrine catalyzed by soybean lipoxygenase in the presence of hydrogen peroxide

A hypothesis that lipoxygenase may mediate N-dealkylation of xenobiotics was investigated using the prototype drug aminopyrine and soybean lipoxygenase as a model enzyme in the presence of hydrogen peroxide. Formaldehyde production as a result of N-demethylation of aminopyrine exhibited pH optimum of 6.5. The reaction was dependent on the incubation time, amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. Under the experimental conditions employed, the specific activity for N-demethylation of aminopyrine was found to be 823 ± 93 nmoles per min/mg protein or 89 ± 10 nmoles per min/nmole of enzyme. The reaction was significantly inhibited by nordihydroguaiaretic acid and gossypol, the classical inhibitors of lipoxygenase. Spectrophotometric analyses indicated the generation of a nitrogen-centered free-radical cation as the initial oxidation product of aminopyrine. The rate of accumulation of this radical species was also dependent on pH, the amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. The radical production was markedly suppressed by ascorbate, glutathione, and dithiothreitol in a concentration-dependent manner. Preliminary data gathered for the oxidation of other chemicals indicated that the lipoxygenase exhibits a unique substrate specificity. Collectively, the evidence presented suggests for the first time that lipoxygenase pathway may be involved in N-demethylation of aminopyrine and other chemicals. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 175–183, 1998     

Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.     

Sucrose Phosphate Synthetase from Various Plant Origins

Some properties of sucrose-P synthetases obtained from various plant tissues, including sweet potato roots, potato tubers and leaves of barley, rape and ladino clover were studied. The specific enzyme activity of the sucrose-P synthetase from sweet potato roots was much lower than that of the sucrose synthetase of the other tissues. The enzyme activity decreased gradually as the roots developed. The optimum pH did not differ between enzyme preparations from sweet potato roots and barley leaves. Manganese chloride exhibited a marked stimulative effect on the sucrose-P synthetase from sweet potato roots and potato tubers, whereas it was inhibited the barley leaf enzyme.

Kinetic studies of sucrose-P synthetase showed that the behavior of the enzyme to the substrates did not differ in the enzyme sources examined. The substrate saturation curve of the enzyme with respect to fructose-6-P was sigmodal in shape, giving a straight line with a slope of 1.35~1.5 (n value) in a plot of the data using the empirical Hill equation. On the other hand, enzymes from all the various tissues exhibited a hyperbolic substrate saturation curve for UDP-glucose, obeying the ordinary Michaelis-Menten type reaction. Manganese chloride had no effect on the Km for UDP-glucose, the S_0.5 for fructose-6-P and the n value of the enzyme from potato tuber tissues.     

Lipoxygenase from potato tubers. Partial purification and properties of an enzyme that specifically oxygenates the 9-position of linoleic acid

A lipoxygenase (EC 1.13.1.13) was partially purified from potato tubers and was shown to differ from previously characterized soya-bean lipoxygenases in the positional specificity and pH characteristics of the oxygenation reaction. The potato enzyme converted linoleic acid almost exclusively (95%) into 9-d-hydroperoxyoctadeca-trans-10,cis-12-dienoic acid. The 13-hydroperoxy isomer was only a minor product (5%). Linolenic acid was an equally effective substrate, which was also oxygenated specifically at the 9-position. The enzyme had a pH optimum at 5.5-6.0 and was inactive at pH9.0. A half-maximal velocity was obtained at a linoleic acid concentration of 0.1mm. No inhibition was observed with EDTA (1mm) and cyanide (1mm) or with p-chloromercuribenzoate (0.2mm). Haemoproteins were not involved in the lipoxygenase activity. The molecular weight of the enzyme was estimated from gel filtration to be approx. 10(5). Preliminary evidence suggested that the enzyme oxygenated the n-10 position of fatty acids containing a penta(n-3, n-6)diene structure.     

Molecular Asymmetry in Pigeonpea Urease: pH Inactivation Studies

Pigeonpea (Cajanus cajan) urease was inactivated by incubating it in buffer of low pHs i.e., 4.8 and 4.5. The pattern of inactivation at both pHs was found to be biphasic, in which half of the activity was destroyed more rapidly than the remaining half. This distribution of active site into two categories is suggestive of site-site heterogeneity, or more specifically, the half-site reactivity of the enzyme moiety. Our pH studies on the rate of reaction showed the presence of two ionizable groups of pK _a values 6.2 ± 0.1 and 8.8 ± 0.1, respectively (Srivastava PK & Kayastha AM, J Mol Catal B: Enz, 16 (2001) 81-89). The later group corresponds to the pK _a value of cysteine group. Here we correlate the loss of urease activity by low pH treatment is due to the effect on essential thiol residues.     

Inhibition of phytoalexin synthesis in arachidonic Acid-stressed potato tissue by inhibitors of lipoxygenase and cyanide-resistant respiration

Arachidonic acid-stressed potato tuber discs synthesized the phytoalexin rishitin. This synthesis was inhibited by salicylhydroxamic acid (SHAM), and to a lesser extent by tetraethylthiuram disulfide (disulfiram). Disulfiram was less effective apparently because it was inactivated in the tuber discs. Disulfiram and SHAM both inhibited cyanide-resistant respiration of whole potato discs and lipoxygenase extracted from these discs. When low disulfiram concentrations were used, the lipoxygenase inhibition was quickly overcome, again because the disulfiram apparently was inactivated by oxidation.     

Phenylethanolamine-N-methyltransferase:alcohol and the mechanism of action

Mixed-solvent systems of methanol and other alcohols and water were used to study the properties of bovine phenylethanolamine-N-methyltransferase. The presence of methanol decreased the binding affinity of the enzyme for its amine substrate but did not alter the maximum velocity. The change in binding was accompanied by an alkaline shift in the pK of an ionizable group in the active site. The well-known property of enzyme inhibition by substrate was also alleviated. Increasing the pH of the medium, in the presence or absence of methanol, increased the maximum velocity but did not alter substrate inhibition. It is proposed that substrate inhibition is due in part to the ionic state of a single unidentified ionizable group in the active site of the enzyme and to a slow release of product. Evidence that an essential, pH-dependent sulfhydryl modulates product release is presented. The properties of phenylethanolamine-N-methyl-transferase are quite responsive to changes in pH, ionic strength, and water content so that the enzyme may well be regulated at the microenvironmental level.     

Protein oxidation in plant mitochondria detected as oxidized tryptophan

The formation of N-formylkynurenine by dioxygenation of tryptophan was detected in peptides from rice leaf and potato tuber mitochondria. Proteins in matrix and membrane fractions were separated by two-dimensional gel electrophoresis and identified using a Q-TOF mass spectrometer. N-Formylkynurenine was detected in 29 peptides representing 17 different proteins. With one exception, the oxidation-sensitive aconitase, all of these proteins were either redox active themselves or subunits in redox-active enzyme complexes. The same site was modified in (i) several adjacent spots containing the P protein of the glycine decarboxylase complex, (ii) two different isoforms of the mitochondrial processing peptidase in complex III, and (iii) the same tryptophan residues in Mn-superoxide dismutase in both rice and potato mitochondria. This indicates that Trp oxidation is a selective process.     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

Purification and properties of a nicotinamide adenine dinucleotide-linked cyclohexanol dehydrogenase from aNocardia species

An inducible pyridine nucleotide-linked cyclohexanol dehydrogenase activity was present in crude extracts from aNocardia species following growth on cyclohexane. The enzyme was purified 126-fold by affinity chromatography and has an oligomeric molecular weight of 145,000 ±5,000. There was an absolute requirement for NAD for activity and the products of the dehydrogenase reaction were stoichiometric amounts of NADH and cyclohexanone. The enzyme had a broad specificity for secondary alcohols including straight-chain secondary alcohols, cyclic and substituted cyclic alcohols, and cyclohexane diols. The apparentK _m values for cyclohexanol and NAD were 3.7×10⁻⁵ M and 2.4×10⁻⁵ M, respectively, and the optimal pH for cyclohexanol oxidation was 10.5. The enzyme was heat sensitive, losing about 50% activity after a 1-min incubation at 45°C. Enzyme activity was completely inhibited by the thiol agent,p-chloromercuribenzoate but not by metal chelating agents.