Hydroxycinnamates in suberin formation

Hydroxycinnamates are found associated with suberin in several forms: covalently linked to the aliphatic suberin; in the residue after suberin-removal; and in the non-polar extractives of suberized tissues in the form of alkyl ferulates. Suberin-associated hydroxycinnamates have been found mainly as ferulic acid-derivatives, sometimes as feruloylamides and in a lesser extent as caffeates. Ferulic acid esters of long-chain ω-hydroxyacids are prevalent in the partial depolymerisation products of suberin. Also, enzymes able to catalyze the feruloylation of ω-hydroxyacids were found timely-associated with the suberization process. It is proposed that ferulic acid, and its dimers, through esterification to ω-hydroxyacids, covalently link the suberin aliphatic polyester to suberin-associated polyaromatics. In this case, the known role of ferulates, and related hydroxycinnamates, as cross-linkers of structurally different polymers would be enlarged to suberized cell-walls.     

Alkyl ferulates in wound healing potato tubers

Seven ferulic acid esters of 1-alkanols ranging in carbon length from C16 to C28 were synthesized and an HPLC protocol for their separation developed. Extracts prepared from wound healing potato (Solanum tuberosum) tubers and analysed by HPLC indicated that alkyl ferulate esters begin to accumulate 3-7 days after wound treatment. Of the nine esters identified by EIMS, (including two esters of odd chain length alkanols) hexadecyl and octadecyl ferulates were predominant. Alkyl ferulate esters were restricted to the wound periderm.     

Continuous synthesis of alkyl ferulate by immobilized Candida antarctica lipase at high temperature

1-Pentyl, 1-hexyl and 1-heptyl ferulates were continuously synthesized at 60–90°C using a reactor system in which a column packed with ferulic acid powders and another column packed with immobilized Candida antarctica lipase particles were connected in series. Conversions greater than 0.9 were achieved for the synthesis of the 1-hexyl and 1-heptyl ferulates at 90°C. The system could be stably operated for the 1-heptyl ferulate synthesis at 90°C for at least two weeks.     

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).     

Kinetics of peroxidation of linoleic acid incorporated into DPPC vesicles initiated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride

In a previous work [Chem. Phys. Lipids 2000 104, 49], we have derived the following rate law for the oxidation of lipids in compartmentalized systems: R(T)=(k(1)/k(t))(0.5) k(p) [In](0.5) c(0.5) [LH], where, R(T) is the total rate of oxidation, k(1) is the rate constant for the production of free radicals, k(t) and k(p) are the intra-particle rate constants for the termination and propagation sets, respectively, [In] is the concentration of a water-soluble initiator, c is the concentration of particles, and [LH] is the intra-particle concentration of oxidable lipid. In the present work, we have investigated on the applicability of the proposed kinetic rate law for a system where it takes place the oxidation of a reactive lipid incorporated into an inert matrix. With this purpose, we have measured the rate of oxidation of linoleic acid incorporated into dipalmitoylphosphatidylcholine vesicles initiated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride as a function of the initiator, particles, and intra-particle LH concentrations. The experimentally determined kinetic orders obtained were 0.54+/-0.02, 0.48+/-0.05 and 0.83+/-0.04 for the dependence of the oxidation rate with initiator, particles, and LH intra-particle concentrations, respectively, in agreement with those theoretically predicted. The lower value obtained for the kinetic order in LH is attributed to a change in k(t) with the increase in oxidable lipid intra-particle concentration. The main point to be emphazised from the results here obtained is that the kinetic rate law for the oxidation of lipids in compartmentalized systems can be significantly different than that observed when to the oxidation takes place in homogeneous solution.     

Physicochemical and biological processes affecting the recovery of exogenously applied ferulic acid from tropical forest soils

Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is found in both plants and soils, and some evidence suggests its involvement in biochemical interactions between plants (allelopathy) and other organisms living in the soil. Knowledge of the processes affecting the concentrations of such potential allelochemicals in soil is essential if we are to understand their roles in the soil environment. It was the intent of this study to address the effects that soil physicochemical and biological processes have on the recovery of exogenously applied ferulic acid from tropical forest soils. Soil extractants used in this study are thought to recover potentially bioavailable concentrations of applied ferulic acid. Water and sodium acetate extractions of soil (immediately and after one and two days) were employed in the recovery of ferulic acid (added at a rate of 5.15 mmoles kg^–1) from steam-sterilized and non-sterilized forest soil materials. Sterilization of soil was used to isolate physicochemical effects from microbial effects on ferulic acid. Results indicate some sterilization treatment effects on the immediate recovery of ferulic acid. Physicochemical and biological processes of soils decreased the recovery of ferulic acid. The immediate recovery of ferulic acid from non-sterile soils is inversely related to the % organic carbon present in the soils. Certain soils have the ability to trap ferulic acid molecules for subsequent release into the soil-solution phase. Furthermore, results suggest that microbial degradation of ferulic acid may only occur in the solution (bulk) phase; ferulic acid molecules thought to be bound to soil surfaces appear to be protected from degradation.Use of trade names in this publication does not imply endorsement by the Organization for Tropical Studies, North Carolina State University or the Savannah River Ecology Laboratory of the products named nor criticism of similar ones not mentioned.     

Kinetics of manganese lipoxygenase with a catalytic mononuclear redox center

Manganese lipoxygenase was isolated from the take-all fungus, Gaeumannomyces graminis, and the oxygenation mechanism was investigated. A kinetic isotope effect, k(H)/k(D) = 21-24, was observed with [U-(2)H]linoleic acid as a substrate. The relative biosynthesis of (11S)-hydroperoxylinoleate (11S-HPODE) and (13R)-hydroperoxylinoleate (13R-HPODE) was pH-dependent and changed by [U-(2)H]linoleic acid. Stopped-flow kinetic traces of linoleic and alpha-linolenic acids indicated catalytic lag times of approximately 45 ms, which were followed by bursts of enzyme activity for approximately 60 ms and then by steady state (k(cat) approximately 26 and approximately 47 s(-1), respectively). 11S-HPODE was isomerized by manganese lipoxygenase to 13R-HPODE and formed from linoleic acid at the same rates (k(cat) 7-9 s(-1)). Catalysis was accompanied by collisional quenching of the long wavelength fluorescence (640-685 nm) by fatty acid substrates and 13R-HPODE. Electron paramagnetic resonance (EPR) of native manganese lipoxygenase showed weak 6-fold hyperfine splitting superimposed on a broad resonance indicating two populations of Mn(II) bound to protein. The addition of linoleic acid decreased both components, and denaturation of the lipoxygenase liberated approximately 0.8 Mn(2+) atoms/lipoxygenase molecule. These observations are consistent with a mononuclear Mn(II) center in the native state, which is converted during catalysis to an EPR silent Mn(III) state. We propose that manganese lipoxygenase has kinetic and redox properties similar to iron lipoxygenases.     

Isomerization increases the postprandial oxidation of linoleic acid but not alpha-linolenic acid in men

Human lipid intake contains various amounts of trans fatty acids. Refined vegetable and frying oils, rich in linoleic acid and/or alpha-linolenic acid, are the main dietary sources of trans-18:2 and trans-18:3 fatty acids. The aim of the present study was to compare the oxidation of linoleic acid, alpha-linolenic acid, and their major trans isomers in human volunteers. For that purpose, TG, each containing two molecules of [1-(13)C]linoleic acid, alpha-[1-(13)C]linolenic acid, [1-(13)C]-9cis,12trans-18:2, or [1-(13)C]-9cis,12cis,15trans-18:3, were synthesized. Eight healthy young men ingested labeled TG mixed with 30 g of olive oil. Total CO(2) production and (13)CO(2) excretion were determined over 48 h. The pattern of oxidation was similar for the four fatty acids, with a peak at 8 h and a return to baseline at 24 h. Cumulative oxidation over 8 h of linoleic acid, 9cis,12trans-18:2, alpha-linolenic acid, and 9cis,12cis,15trans-18:3 were, respectively, 14.0 +/- 4.1%, 24.7 +/- 6.7%, 23.6 +/- 3.3%, and 23.4 +/- 3.7% of the oral load, showing that isomerization increases the postprandial oxidation of linoleic acid but not alpha-linolenic acid in men.     

Biochemical characterization of the dual positional specific maize lipoxygenase and the dependence of lagging and initial burst phenomenon on pH, substrate, and detergent during pre-steady state kinetics

The wound-inducible lipoxygenase obtained from maize is one of the nontraditional lipoxygenases that possess dual positional specificity. In this paper, we provide our results on the determination and comparison of the kinetic constants of the maize lipoxygenase, with or without detergents in the steady state, and characterization of the dependence of the kinetic lag phase or initial burst, on pH, substrate, and detergent in the pre-steady state of the lipoxygenase reaction. The oxidation of linoleic acid showed a typical lag phase in the pre-steady state of the lipoxygenase reaction at pH 7.5 in the presence of 0.25% Tween-20 detergent. The reciprocal correlation between the induction period and the enzyme level indicated that this lag phenomenon was attributable to the slow oxidative activation of Fe (II) to Fe (III) at the active site of the enzyme as observed in other lipoxygenase reactions. Contrary to the lagging phenomenon observed at pH 7.5 in the presence of Tween-20, a unique initial burst was observed at pH 6.2 in the absence of detergents. To our knowledge, the initial burst in the oxidation of linoleic acid at pH 6.2 is the first observation in the lipoxygenase reaction. Kinetic constants (K(m) and k(cat) values) were largely dependent on the presence of detergent. An inverse correlation of the initial burst period with enzyme levels and interpretations on kinetic constants suggested that the observed initial burst in the oxidation of linoleic acid could be due to the availability of free fatty acids as substrates for binding with the lipoxygenase enzyme.     

Modification of esterified cell wall phenolics increases vulnerability of tall fescue to herbivory by the fall armyworm

Feruloylation of arabinoxylan in grass cell walls leads to cross-linked xylans. Such cross-linking appears to play a role in plant resistance to pathogens and insect herbivores. In this study, we investigated the effect of ferulate cross-linking on resistance to herbivory by fall armyworm (Spodoptera frugiperda) making use of genetically modified tall fescue [Schedonorus arundinaceus (Festuca arundinacea)] expressing a ferulic acid esterase gene. Mature leaves of these plants have significant reduced levels of cell wall ferulates and diferulates but no change in acid detergent lignin. These reduced levels of esterified cell wall ferulates in transgenic plants had a positive effect on all measures of armyworm larval performance examined. More larvae survived (89 vs. 57?%) and grew faster (pupated 2.1?days sooner) when fed transgenic leaves with reduced levels of cell wall ferulates, than when fed control tall fescue leaves where levels of cell wall ferulates were not altered. Overall, mortality, growth and food utilization were negatively associated with level of esterified cell wall ferulates and diferulates in leaves they were fed. This study is the first to use transgenic plants with modified level of cell wall esterified ferulates to test the role of feruloylation in plant resistance to insects. It is concluded that the accumulation of ferulates and the cross-linking of arabinoxylans via diferulate esters in the leaves of tall fescue underlies the physical barrier to insect herbivory. Reducing ferulate cross-linking in grass cell walls could increase susceptibility of these plants to insect folivores.     

The feruloyl esterase system of Talaromyces stipitatus: determining the hydrolytic and synthetic specificity of TsFaeC

The active site of the recombinant Talaromyces stipitatus type-C feruloyl esterase (TsFaeC) was probed using a series of C1-C4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of k(cat) and k(cat)/K(m) values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl group transfer to l-arabinose, using as a reaction system a ternary water-organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.     

Role of phospholipids in the regulation of activity of porcine leukocyte 12-lipoxygenase]

12-Lipoxygenase from porcine leukocytes was partially purified by using of DEAE-Toyopearl chromatography (pH 7.5). Phosphatidylcholine and Phosphatidylinositol in reaction mixtures with mixed micelles Lubrol PX/linoleic acid inhibited the enzyme. The pH-optimum of lipoxygenase reaction in presence of phospholipids shifted into alkaline region. In the absence of phospholipids 3 additional substrate molecules bound with enzyme-substrate complex. In the presence of either phosphatidylcholine of phosphatidylinositol up to 2 substrate molecules bound with enzyme-substrate complex. The phospholipids competed with linoleic acid for one of the enzyme binding centers. A kinetic scheme of 12-lipoxygenase reaction has been proposed: Phosphatidylinositol lowered the values of Ks and Kns of the reaction of linoleic acid oxidation by 12-lipoxygenase, while phosphatidylcholine had opposite effect on these parameters. We suppose that phospholipids can regulate 12-lipoxygenase activity via control of the enzyme affinity to the substrate (polyunsaturated fatty acid).     

Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila

A ferulic acid esterase (FAE) from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile), belonging to the carbohydrate esterase family 1 (CE-1), was functionally expressed in methylotrophic yeast Pichia pastoris. The putative FAE from the genomic DNA was successfully cloned in P. pastoris X-33 to confirm that the enzyme exhibits FAE activity. The recombinant FAE was purified to its homogeneity (39 kDa) and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme shows a preference for the hydrolysis of methyl caffeate and p-coumarate and a strong preference for the hydrolysis of n-butyl and iso-butyl ferulate. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose, whilst it was found capable of de-esterifying acetylated glucuronoxylans. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with an M3 xylanase from Trichoderma longibrachiatum (a maximum of 41% total FA released after 1 h incubation). Prediction of the secondary structure of MtFae1a was performed in the PSIPRED server whilst modelling the 3D structure was accomplished by the use of the HH 3D structure prediction server.     

Efficient enzymatic preparation of hydroxycinnamates in ionic liquids enhances their antioxidant effect on lipoproteins oxidative modification

Biocatalytic lipophilization of hydroxycinnamic acids was performed in several BF₄^? and PF₆^? imidazolium ionic liquids using immobilized lipases. The influence of various reaction parameters on the performance of the biocatalytic process was pointed out, using as model reaction the esterification of ferulic acid. The biocatalytic lipophilization strongly depended on the ion composition of ionic liquids used. Conversions and initial reaction rates were significantly higher in PF₆^? as compared with BF₄^? ionic liquids and commonly used organic solvents. The high enzyme stability and the relative solubility of substrate versus product in PF₆^? ionic liquids can account for the improved synthesis of lipophilic ferulates. These lipophilic derivatives, when used at a concentration of up to 400-fold lower than the parental compound, efficiently inhibited the oxidation of isolated LDL, HDL and total serum in vitro. Moreover, it has been shown for the first time that the lipophilic ferulates improve the antioxidant efficiency of the HDL_3c towards LDL in vitro oxidation.     

Decomposing potassium peroxychromate produces hydroxyl radical (.OH) that can peroxidize the unsaturated fatty acids of phospholipid dispersions

The unsaturated fatty acyl residues of egg yolk lecithin are selectively removed when bilayer dispersions of the lipid are exposed to decomposing peroxychromate at pH 7.6 or pH 9.0. Mannitol (50 mM or 100 mM)partially prevents the oxidation of the phospholipid due to decomposing peroxychromate at pH 7.6 and the amount of lipid lost is inversely proportional to the concentration of mannitol. N,N-Dimethyl-p-nitrosoaniline, mixed with the lipid in a molar ratio of 1.3:1, completely prevents the oxidation of lipid due to decomposing peroxychromate at pH 9.0, but some linoleic acid is lost if the incubation is done at pH 7.6. If the concentration of this quench reagent is reduced tenfold, oxidation of linoleic acid by decomposing peroxychromate at pH 9.0 is observed. Hydrogen peroxide is capable of oxidizing the unsaturated fatty acids of lecithin dispersions. Catalase or boiled catalase (2 mg/ml) protects the lipid from oxidation due to decomposing peroxychromate at pH 7.6 to approximately the same extent, but their protective effect is believed to be due to the non-specific removal of .OH. It is concluded that .OH is the species responsible for the lipid oxidation caused by decomposing peroxychromate. This is consistent with the observed bleaching of N,N-dimethyl-p-nitrosoanaline and the formation of a characteristic paramagnetic .OH adduct of the spin trap, 5,5-dimethylpyrroline-1-oxide.     

Kinetics of cytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde.

The P450 2E1-catalyzed oxidation of ethanol to acetaldehyde is characterized by a kinetic deuterium isotope effect that increases K(m) with no effect on k(cat), and rate-limiting product release has been proposed to account for the lack of an isotope effect on k(cat) (Bell, L. C., and Guengerich, F. P. (1997) J. Biol. Chem. 272, 29643-29651). Acetaldehyde is also a substrate for P450 2E1 oxidation to acetic acid, and k(cat)/K(m) for this reaction is at least 1 order of magnitude greater than that for ethanol oxidation to acetaldehyde. Acetic acid accounts for 90% of the products generated from ethanol in a 10-min reaction, and the contribution of this second oxidation has been overlooked in many previous studies. The noncompetitive intermolecular kinetic hydrogen isotope effects on acetaldehyde oxidation to acetic acid ((H)(k(cat)/K(m))/(D)(k(cat)/K(m)) = 4.5, and (D)k(cat) = 1.5) are comparable with the isotope effects typically observed for ethanol oxidation to acetaldehyde, and k(cat) is similar for both reactions, suggesting a possible common catalytic mechanism. Rapid quench kinetic experiments indicate that acetic acid is formed rapidly from added acetaldehyde (approximately 450 min(-1)) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, approximately 90% of the acetaldehyde intermediate is directly converted to acetic acid without dissociation from the enzyme active site. Competition experiments suggest that P450 2E1 binds acetic acid and acetaldehyde with relatively high K(d) values, which preclude simple tight binding as an explanation for rate-limiting product release. The existence of a rate-determining step between product formation and release is postulated. Also proposed is a conformational change in P450 2E1 occurring during the course of oxidation and the discrimination of P450 2E1 between acetaldehyde and its hydrated form, the gem-diol. This multistep P450 reaction is characterized by kinetic control of individual reaction steps and by loose binding of all ligands.     

Characteristics of the aggregative state of the substrate in the reaction of 5-lipoxygenase oxidation of linoleic acid

5-lipoxygenase (EC 1.13.11.12) oxidizes polyunsaturated fatty acids by molecular oxygen. The enzyme acts in close contact with the cell membranes, which main components are ionic and non-ionic lipids. In order to investigate the kinetic parameters of 5-lipoxygenase reaction in vitro, extremely hydrophobic fatty acid substrate (linoleic acid) should be solubilized in the reaction mixture. We used Lubrol PX ("Sigma" Chem. Co), as a non-ionic detergent consisted of oligoethylene glycol and fatty alcohol. Linoleic acid and Lubrol PX formed mixed micelles thus solubilizing the fatty acid substrate in a buffer with appropriate pH. We have studied the sizes and shapes of mixed micelles Lubrol PX/linoleic acid (aggregates type 1) and Lubrol PX/linoleic acid/SDS (aggregates type 2; SDS was an effective activator of potato tuber 5-lipoxygenase) by means of gel-filtration and laser light scattering techniques. The parameters under investigation were molecular weights, Stocks radii and shapes of the mixed micelles. The average molecular weights and Stocks radii of the mixed micelles type 1 determined by mean of gel-filtration on Sephadex G-200 were 95,142 +/- 5184 Da and 3.45 +/- 0.11 nm, respectively. The same parameters for the mixed micelles type 2 were 73,694 +/- 893 Da and 3.02 +/- 0.02 nm, respectively. The strong similarity in physicochemical parameters for both types of mixed micelles indicated that SDS did not influence the size and shape of mixed micelles of Lubrol PX and linoleic acid. The activatory action of SDS on potato tuber lipoxygenase may be a result of electrostatic effect or direct participation of SDS in enzymatic catalysis. The laser light scattering technique allowed to determine two main fraction of particles in type 1 system with hydrodynamic diameters 2.6 and 5.7 nm and relative contribution to light scattering 13 and 87%, respectively. The particles with d = 5.7 nm were interpreted as the mixed micelles. The particles with d = 2.6 nm were interpreted as isolated molecules of Lubrol PX, linoleic acid and (or) their premicellar aggregates. The data obtained are to be used in creation of reliable physical and mathematical models of 5-lipoxygenase.     

Cell wall oxalate oxidase modifies the ferulate metabolism in cell walls of wheat shoots

Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.     

Involvement of a flavosemiquinone in the enzymatic oxidation of nitroalkanes catalyzed by 2-nitropropane dioxygenase

2-Nitropropane dioxygenase (EC 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite. In this study, the ncd-2 gene encoding for the enzyme in Neurospora crassa was cloned, expressed in Escherichia coli, and the resulting enzyme was purified. Size exclusion chromatography, heat denaturation, and mass spectroscopic analyses showed that 2-nitropropane dioxygenase is a homodimer of 80 kDa, containing a mole of non-covalently bound FMN per mole of subunit, and is devoid of iron. With neutral nitroalkanes and anionic nitronates other than propyl-1- and propyl-2-nitronate, for which a non-enzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site. The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant k(cat)/K(m) determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons. The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the carbonyl product. Enzyme-monitored turnover with ethyl nitronate as substrate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not molecular oxygen, as suggested by anaerobic substrate reduction with nitroethane or ethyl nitronate. Substrate deuterium kinetic isotope effects with 1,2-[(2)H(4)]nitroethane and 1,1,2-[(2)H(3) ethyl nitronate at pH 8 yielded normal and inverse effects on the k(cat)/K(m) value, respectively, and were negligible on the k(cat) value. The k(cat)/K(m) and k(cat) pH profiles with anionic nitronates showed the requirement of an acid, whereas those for neutral nitroalkanes were consistent with the involvement of both an acid and a base in catalysis. The kinetic data reported herein are consistent with an oxidasestyle catalytic mechanism for 2-nitropropane dioxygenase, in which the flavin-mediated oxidation of the anionic nitronates or neutral nitroalkanes and the subsequent oxidation of the enzyme-bound flavin occur in two independent steps.     

Kinetic mechanisms of linoleic acid oxidation by 5-lipoxygenase from Solanum tuberosum L

Linoleic acid oxidation by 5-lipoxygenase from Solanum tuberosum has been studied as affected by sodium dodecylsulfate (Ds-Na). The reaction system consisted of 5-lipoxygenase and mixed micelles of linoleic acid and Lubrol PX. It contained varying amounts of the enzyme effector--Ds-Na. The enzyme showed a pronounced cooperativity, and the reaction was governed by the Hill equation with h = 3.7. On the other side, increasing amounts of Ds-Na added to the system caused a tremendous increase of enzyme activity and simultaneous decline of h, with was proportional to Ds-Na concentration. Ds-Na had dual effect on 5-lipoxygenase--there was an optimal concentration of the compound (0.34 mM Lubrol PX; 0.2 mM LA; 0.13 mM Ds-Na; pH = 6.3) causing the 4-fold highest activation and h = 1.6. The further increase of Ds-Na led to the enzyme inhibition. If Ds-Na was 0.5 mM, h became 1. At this point, each molecule of 5-lipoxygenase bound 3 molecules of Ds-Na and 1 molecule of linoleic acid, thus the total number of occupied binding sites was 4. A kinetic scheme of 5-lipoxygenase reaction has been proposed. It was found that the enzyme's kinetic behaviour could be explaine if assumed an existence of a special noncatalytic binding centre capable of binding several (up to 3) molecules of either substrate, or effector. Such a centre can serve as an anchoring site facilitating the enzyme binding to the surface of lipid aggregates containing insolubilized substrate molecules. Replacing linoleic acid in the binding site, Ds-Na activates the enzyme, possibly due to the much more effective translocation of 5-lipoxygenase to the surface of lipid aggregates. This mechanism can be an universal alternative to the FLAP-type regulation of 5-lipoxygenase activities.