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1.
Methiocarb, an aromatic-alkyl sulfide insecticide was enzymatically oxidized into its sulfoxide by microsomes from soybean cotyledons. No further oxidation into sulfone was detected. Distribution of the sulfoxidase activity was studied in soybean seedlings and found maximal in cotyledons. Subcellular fractionation of cotyledons homogenates indicated that the activity was almost entirely associated with the microsomal fraction. Sulfoxidation of methiocarb did not require cofactors such as NAD(P)H. Nevertheless, the sulfoxidase did not act as a peroxidase. 相似文献
2.
T. Buronfosse P. Moroni E. Beno
t J. L. Rivire 《Journal of biochemical and molecular toxicology》1995,10(4):179-189
Evidence based on thermal lability and enzyme inhibition data suggests that the sulfoxidation of methiocarb (an N-methylcarbamate insecticide) by rat liver microsomes is catalyzed by flavin-containing monooxygenase(s) (FMO) and by cytochrome(s) P450 (P450). In control rats, the relative proportion is ca. 50% P450:50% FMO. Stereoselective formation of methiocarb sulfoxide from the corresponding sulfide has also been examined to compare the enantioselectivity of the two different enzyme systems. Only the FMO-dependent sulfoxidation presents a high stereoselectivity with an enantiomeric excess of 88% in favor of the (A)-enantiomer. Pretreatment of rats with different P450 inducers such as phenobarbital, 3-methylcholanthrene, dexamethasone, and pyrazole did not affect, or decreased, the rate of methiocarb sulfoxidation. Stereoselectivity of the reaction was modified, mainly because of changes in the relative involvement of FMO and P450 in sulfoxidase activity in pretreated animals. The acetylcholinesterase inhibition properties of methiocarb and its main metabolites were also investigated. Racemic methiocarb sulfoxide was slightly less inhibitory (Ki = 0.216 μM?1· min?1) than methiocarb, but a 10-fold difference was observed between the bimolecular rate constants found for the two sulfoxides produced (0.054 and 0.502 μM?1·min?1 for the (A) and (B) enantiomers, respectively). © 1995 John Wiley & Sons, Inc. 相似文献
3.
Increased NADPH-dependent chemiluminescence by microsomes after chronic ethanol consumption 总被引:1,自引:0,他引:1
The generation of reactive oxygen intermediates by microsomes from ethanol-fed rats and pair-fed controls was determined by assaying for NADPH-dependent chemiluminescence. In the absence or presence of added ferric complexes, microsomal light emission was elevated several-fold after chronic ethanol consumption. Iron complexes such as ferric-citrate or ferric-ATP stimulated, while ferric-EDTA, inhibited microsomal chemiluminescence. Freeze-thawing the microsomes to elevate their content of lipid hydroperoxides resulted in large increases in chemiluminescence; under all conditions, the light emission remained several-fold higher with microsomes from the ethanol-fed rats. Chemiluminescence was not sensitive to superoxide dismutase, catalase, or the hydroxyl radical scavenging agent, dimethyl sulfoxide, but was inhibited by antioxidants and by glutathione. Replacing air with a mixture of 50% nitrogen-50% air or 50% carbon monoxide-50% air had no effect on chemiluminescence by microsomes from the pair-fed controls. However, the chemiluminescent response by microsomes from the ethanol-fed rats was inhibited about 50% by the nitrogen mixture, and was further inhibited (about 75% of values found with 100% air, and 50% of values found with 50% nitrogen-50% air) with the carbon monoxide mixture. The sensitivity to carbon monoxide suggests the possibility that the alcohol-inducible cytochrome P-450 isozyme may contribute, in part, to the elevated light emission produced by microsomes from the ethanol-fed rats. The increase in chemiluminescence by microsomes after chronic ethanol consumption appears to reflect an elevated level of lipid hydroperoxides as well as an increased rate of generation of reactive oxygen species. 相似文献
4.
The generation of active oxygen species by microsomes isolated from soybean seedlings was studied. NADPH-dependent superoxide anion production was 5.0 ± 0.4 nmol · min−1 mg−1 of microsomal protein. Hydrogen peroxide generation by microsomes was 1.40 ± 0.05 nmol · min−1 mg−1 of protein. Hydroxyl radical production, in the presence of ferric EDTA, evaluated through the generation of formaldehyde from dimethyl sulfoxide or tert-butyl alcohol was 0.50 ± 0.04 and 0.44 ± 0.03 nmol · min−1 mg−1, respectively. NADH proved to be suitable as cofactor for oxygen radical generation by microsomes from soybean seedlings. Because transition metals are implicated in radical generation by biological systems, the ability of microsomal membranes to reduce iron complexes was studied. Ferric ATP, ferric citrate, ferric ADP, ferric diethylenetriamine pentaacetic acid, and ferric EDTA were efficiently reduced in the presence of either NADPH or NADH as cofactor. The pattern of effectiveness of the different ferric complexes, on superoxide anion, hydrogen peroxide, and hydroxyl radical production, was similar to that found with animal microsomes. The data presented here indicate that microsomal ability to catalyze oxygen radical generation must be considered as an important contribution to cellular radical steady-state concentrations in cells from soybean seedlings. 相似文献
5.
S L Ripp K Itagaki R M Philpot A A Elfarra 《Archives of biochemistry and biophysics》1999,367(2):322-332
Methionine has previously been shown to be S-oxidized by flavin-containing monooxygenase (FMO) forms 1, 2, and 3. The most efficient catalyst was FMO3, which has a Km value for methionine S-oxidation of approximately 4 mM, and exhibits high selectivity for formation of the d-diastereoisomer of methionine sulfoxide. The current studies provide evidence for an additional methionine S-oxidase activity in liver microsomes. Human and rabbit liver microsomes exhibited a biphasic response to methionine at concentrations ranging from 0.05 to 10 mM, as indicated by both Eadie-Hofstee plots and nonlinear regression. The low-affinity component of the biphasic response had Km values of approximately 3 and 5 mM for humans and rabbits, respectively, as well as high diastereoselectivity for methionine sulfoxide formation. The low-affinity activity in rabbit liver microsomes was inhibited by methimazole, S-allyl-l-cysteine, and by mild heat treatment, suggesting the activity is FMO3. The high-affinity component of the biphasic response had Km values of approximately 0.07 and 0.04 mM for humans and rabbits, respectively, as well as lower diastereoselectivity for methionine sulfoxide formation. Further characterization of the high-affinity activity in rabbit liver microsomes indicated lack of involvement of cytochrome P450s or reactive oxygen species. The high-affinity activity was inhibited 25% by potassium cyanide and greater than 50% by methimazole and S-allyl-l-cysteine. Mild heat treatment produced 85% inhibition of the low-affinity activity, but only 30% inhibition of the high-affinity activity. Both high- and low-affinity activities were decreased by 85% in flavin-depleted microsomes. Because these results suggested the additional S-oxidase activity has characteristics of an FMO, recombinant human FMO4 was evaluated as a potential catalyst of this activity. Recombinant FMO4 catalyzed S-oxidation of both methionine and S-allyl-l-cysteine, with similar diastereoselectivity to the high-affinity microsomal S-oxidase; however, the Km values for both reactions appeared to be greater than 10 mM. In summary, these studies provide evidence for two microsomal methionine S-oxidase activities. FMO3 is the predominant catalyst at millimolar concentrations of methionine. However, at micromolar methionine concentrations, there is an additional S-oxidase activity that is distinct from FMO3. 相似文献
6.
R W Egan P H Gale E M Baptista K L Kennicott W J VandenHeuvel R W Walker P E Fagerness F A Kuehl 《The Journal of biological chemistry》1981,256(14):7352-7361
oxidations of organic sulfides, amines, and even enzymes catalyzed by purified and microsomal forms of prostaglandin cyclooxygenase-hydroperoxidase have been studied using O2 incorporation into arachidonic acid to monitor oxygenase and [14C]15-hydroperoxyprostaglandin E2 reduction to prostaglandin E2 to measure hydroperoxidase. The oxygenase was protected by phenol against the irreversible deactivation induced by low levels of hydroperoxides. Furthermore, the EPR signal noted during reactions with the microsomal enzyme probably reflected the adventitious oxidation of endogenous materials. As described previously for phenol and other reducing cosubstrates, methyl phenyl sulfide (MPS) increased hydroperoxidase activity at all concentrations studied, while stimulating oxygenase at low levels and inhibiting it at 5-10 mM. In stoichiometric equivalence with 15-hydroperoxyprostaglandin E2 reduction, MPS was enzymatically oxidized to its analogous sulfoxide, methylphenyl sulfoxide, acquiring an oxygen atom exclusively from the hydroperoxide and demonstrating some chiral character. In contrast, other oxidizable compounds such as N,N-dimethylphenylenediamine and aminopyrine reacted via radical intermediates. Phenylbutazone, which is oxidized using dissolved molecular oxygen, did not compete with MPS oxidation. Hence, MPS was oxidized while bound to the enzyme, whereas the amine oxidation occurred in solution via an enzyme-formed oxidant. The Soret peak noted with cyclooxygenase-hydroperoxidase was examined as a possible measure of this binding, but was also noted in denatured and deactivated enzyme, suggesting that its relevance should be reconsidered. Despite the similarities in their drug-metabolizing profiles, cyclooxygenase-hydroperoxidase is clearly distinct from cytochrome P-450. The mechanism of this hydroperoxidase is considered in the context of other more extensively studied peroxidases. 相似文献
7.
C S Jewell G W Winston 《Comparative biochemistry and physiology. B, Comparative biochemistry》1989,92(2):329-339
1. The microsomal mixed-function oxygenase (MFO) system from the hepatopancreas and green gland of the red swamp crayfish, Procambarus clarkii has been characterized with respect to the constitution of electron transport proteins and the ability to catalyze the metabolism of xenobiotics. 2. Cytochrome P-450 content of hepatopancreas microsomes was approximately 10-fold higher than that of green gland and comparable to that of rat liver. NADPH-cytochrome c reductase activity in hepatopancreas microsomes was approximately 2% of that found in rat liver microsomes. 3. Green gland microsomes catalyzed higher turnover rates of aminopyrine N-demethylase and benzo[a]pyrene hydroxylase than hepatopancreas microsomes. With hepatopancreas microsomes, organic hydroperoxides supported a greater rate of aminopyrine N-demethylation than did NADPH plus O2. 4. P. clarkii hepatopancreas microsomes generally displayed a lower binding affinity (Ks) for a number of type I and type II ligands than did rat liver microsomes. 相似文献
8.
Detection of in vivo lipid peroxidation using the thiobarbituric acid assay for lipid hydroperoxides 总被引:6,自引:0,他引:6
Thiobarbituric acid (TBA) assays which have been modified for detection of lipid hydroperoxides appear to be useful for demonstration of in vivo lipid peroxidation. Since these methods require heating tissue membranes with the buffered TBA, there is a possibility of interference from the detection of autoxidation that occurs during heating. These studies were undertaken to investigate conditions which favor TBA color production from hydroperoxide while limiting autoxidation during the assay. An acetic acid-sodium acetate buffered (pH 3.6) TBA assay was used. Heating linoleic acid hydroperoxide with 50 microM ferric iron or under nitrogen nearly doubled color production compared to heating it with no added iron or under air. The lipid antioxidant butylated hydroxytoluene inhibited color production from fatty acid hydroperoxides. When tissue fractions, including liver and lung microsomes and lung whole membranes, were heated in the assay, color production was greater under air than under nitrogen and was much greater under oxygen. When liver microsomes from carbon tetrachloride-exposed rats were used, color was increased only when oxygen was present in the heating atmosphere. The results with tissue fractions appear to demonstrate autoxidation during color development rather than the presence of preformed hydroperoxides. Finally, it was found that color production from membrane fractions was dependent on the vitamin E content of the membranes. It appears that autoxidation during heating should be limited by heating under nitrogen and not by adding antioxidants, which inhibit color production from hydroperoxides. As the vitamin E effect demonstrates, antioxidant status must be considered, since a change in color production could result from a change in antioxidant content without the accumulation of lipid hydroperoxides. 相似文献
9.
The microsomal fraction from fish muscle has previously been shown to catalyze the oxidation of its lipid. In this study we have studied the rate of the reaction in the frozen state. The rate was dependent on temperature, decreasing with decreasing temperature. When the microsomes were frozen in the presence of NaCl there was greater activity than when they were frozen in the presence of KCl. The specific activity of the oxidation decreased with increasing protein concentration. This is possibly due to the limitation of oxygen in the frozen system. Lipid oxidation is a complex reaction and both initial products (lipid hydroperoxides) and breakdown products (those reacting with malondialdehyde) were measured. This ratio was relatively constant over a variety of conditions indicating that the rate-limiting step of the reaction occurred prior to the formation of lipid hydroperoxide. A study of the reaction at above-freezing temperatures and below-freezing temperatures in the presence of miscible solvents to prevent freezing at temperatures below 0 °C gave results which were consistent with the hypothesis that ice crystal formation had an accelerating effect on the reaction. Presumably this is due to concentration of reactants since freezing and thawing of the microsomes did not affect their rates of lipid oxidation. Potent inhibitors of the lipid oxidation reaction were found in the soluble fraction of the muscle tissue. These were both high-molecular and low-molecular-weight compounds. The low-molecular-weight inhibitors were more effective in the frozen state while the high-molecular-weight compounds were relatively more effective in the reaction catalyzed at temperatures above freezing. 相似文献
10.
There exist at least two kinds of CO-binding hemoproteins in microsomal fractions of germinating pea (Pisum sativum) seeds. One of them is cytochrome P-450 and the other is also a protoheme protein (judged from its pyridine hemochrome spectrum), which is not hitherto reported. The content of the new hemoprotein is much higher than that of cytochrome P-450 in the early stage of germination. During germination the former decreases and the latter increases. The new hemoprotein is not appreciably reduced by sodium dithionite alone within a few minutes, but, it is easily reduced by dithionite in the presence of methyl viologen and also by hydrogen peroxide when CO is present. The addition of hydrogen peroxide to pea microsomes in the absence of CO causes destruction of the hemoprotein and also decolorization of endogenous carotenoid. Destruction of these components is brought about by organic hydroperoxides independently of the presence of CO. In the presence of hydroxylamine, the addition of hydroperoxides to the microsomes results in the formation of an absorption spectrum similar to the spectra of ferrous-NO complexes of protoheme proteins. When N,N-dimethyl p-phenylenediamine is present, the reaction of pea microsomes with hydroperoxides gives a spectrum similar to that of the ferryl form of myoglobin. The reactions of the hemoprotein with hydroperoxides are inhibited by alpha,alpha'-dipyridyl and aniline, with which pea microsomes form binding spectra. The microsomes form a rather stable difference spectrum with hydroxylamine. However, the hemoprotein is destroyed when hydroxylamine is added to the microsomes in the reduced state. 相似文献
11.
F F Kadlubar K C Morton D M Ziegler 《Biochemical and biophysical research communications》1973,54(4):1255-1261
Organic hydroperoxides are capable of supporting the C-oxidation of several different amines in the presence of hepatic microsomes. Evidence is presented that indicates that microsomal cytochrome P-450 acts as the catalyst. Removal of the NADPH-cytochrome oxidoreductase or essential phospholipid from microsomes does not significantly affect the peroxidase activity. Of the amine substrates C-oxidized by organic hydroperoxides in the presence of microsomes, only aminopyrine and dimethylaniline are rapidly oxidized by hydroperoxides in the presence of catalase. The catalase-mediated reaction can also be distinguished from the microsomal-catalyzed reaction by the use of differential inhibitors. 相似文献
12.
Microsomal fractions isolated from parsley cell suspension cultures, which had been challenged with an elicitor from either Alternaria carthami or Phytophthora megasperma f. sp. glycinea, catalyzed the formation of psoralen from synthetic [3-14C](+)marmesin. Whereas psoralen was the only product formed in incubations with Alternaria-induced microsomes, another unidentified product was isolated from incubations with Phytophthora-induced microsomes. The latter product is neither a precursor nor a product of psoralen. In contrast, microsomes isolated from non-induced parsley cells lacked both of these catalytic activities. The formation of psoralen depends on NADPH as a cofactor and molecular oxygen. Blue-light-reversible CO inhibition and inhibition by various synthetic chemicals known to bind to cytochromes P450 indicated that the reaction is catalyzed by an elicitor-inducible cytochrome P450-dependent psoralen synthase. Fractionation of microsomal preparations by centrifugation revealed that psoralen synthase is associated with the endoplasmic reticulum. Our results suggest that the endoplasmic reticulum of cultured parsley cells is the primary target in the previously reported differential induction by elicitors from these two non-pathogenic strains of fungi. 相似文献
13.
Nitric oxide decreases superoxide anion generation by microsomes from soybean embryonic axes 总被引:9,自引:0,他引:9
Experiments were carried out to evaluate the effects of exposure to nitric oxide on the ability by NADPH‐dependent microsomal electron transfer to generate oxygen radicals. Such interactions could play a role in the potential antioxidant action of nitric oxide (NO). Isolated microsomes from soybean ( Glycine max [L.] Merr. cv. Hood) embryonic axes were exposed to an exogenously added source of nitric oxide (NO) (S‐nitrosoglutathione + dithiothreitol). The O2 − generation rate by microsomes exposed to NO decreased significantly as compared to the rate measured in microsomes incubated in the absence of NO. The exposure of the microsomes to the NO donor did not alter the microsomal rate of hydroxyl radical generation. Preincubation of the microsomes with the NO donor affected neither iron reduction rate nor activity of cytochrome c reductase. However, cytochrome P450 activity was significantly inhibited after exposure to NO. This inhibition was completely prevented by hemoglobin. The data are consistent with the hypothesis that NO exhibits a potential antioxidant role in the plant cell by decreasing the rate of generation of superoxide anion. Since endogenous NO was detected in homogenates of soybean embryonic axes by EPR studies, this interaction between NO and cytochrome P450 in soybean embryonic axes could be a factor of relevance for the control of oxidative stress in vivo. 相似文献
14.
In vivo administration of acetone influences a variety of reactions catalyzed by rat liver microsomes. The effect of chronic treatment with acetone (1% acetone in the water for 10-12 days) on interaction with iron and subsequent oxygen radical generation by liver microsomes was evaluated. Microsomes from the acetone-treated rats displayed elevated rates of H2O2 generation, an increase in iron-dependent lipid peroxidation, and enhanced chemiluminescence upon the addition of t-butylhydroperoxide. The ferric EDTA-catalyzed production of formaldehyde from DMSO or of ethylene from 2-keto-4-thiomethylbutyrate was increased 2-fold after acetone treatment. This increase in hydroxyl radical generation was accompanied by a corresponding increase in NADPH utilization and was sensitive to inhibition by catalase and a competitive scavenger, ethanol, but not to superoxide dismutase. In vitro addition of acetone to microsomes had no effect on oxygen radical generation. Associated with the chronic acetone treatment was a 2-fold increase in the microsomal content of cytochrome P-450 and in the activity of NADPH-cytochrome-P-450 reductase. It appears that increased oxygen radical generation by microsomes after chronic acetone treatment reflects the increase in the major enzyme components which comprise the mixed-function oxidase system. 相似文献
15.
Dolichol kinase activity in microsomes from etiolated rye seedlings had a pH optimum at 8 with a shoulder at pH 6.5. Triton X-100 (0.4%) was required for optimum activity. Exogenous divalent cations did not enhance activity, although Mg+2 was added routinely. Rye microsomes were found to contain dolichol and polyprenol in a ratio of 3 to 2. Rye, soybean embryo, and rat liver microsomes catalyzed the synthesis of 78, 52, and 516 nmol [14C]dolichyl phosphate/(mg microsomal protein.h) compared with 21, 22, and 49 nmol [3H]polyprenyl phosphate/(mg microsomal protein.h), respectively. It is clear that microsomes from plant systems can catalyze the phosphorylation of polyprenol better than rat liver when compared with their abilities to catalyze the phosphorylation of dolichol. It is not known whether one or more kinases is responsible for catalyzing the phosphorylation of these two closely related groups of compounds. 相似文献
16.
Methionine sulfoxide reductases, enzymes that reverse the oxidation of methionine residues, have been described in a wide
range of species. The reduction of the diastereoisomers of oxidized methionine is catalyzed by two different monomeric methionine
sulfoxide reductases (MsrA and MsrB) and is best understood as an evolutionary response to high levels of oxygen either in
the Earth’s atmosphere or possibly in more localized environments. Phylogenetic analyses of these proteins suggest that their
distribution is the outcome of a complex history including many paralogy and lateral gene transfer events.
Electronic Supplementary Material Electronic Supplementary material is available for this article at
and accessible for authorised users.
[Reviewing Editor: Dr. Martin Kreitman] 相似文献
17.
R W Lambrecht P R Sinclair N Gorman J F Sinclair 《Archives of biochemistry and biophysics》1992,294(2):504-510
Previous work suggested that the oxidation of uroporphyrinogen to uroporphyrin is catalyzed by cytochrome P450IA2. Here we determined whether purified reconstituted mouse P450IA1 and IA2 oxidize uroporphyrinogen. Cytochromes P450IA1 and IA2 were purified from hepatic microsomes from 3-methylcholanthrene (MC)-treated C57BL/6 mice, using a combination of affinity chromatography and high performance liquid chromatography. Reconstituted P450IA1 was more active than P450IA2 in catalyzing ethoxyresorufin-O-deethylase (EROD) activity, whereas P450IA2 was more active than P450IA1 in catalyzing uroporphyrinogen oxidation (UROX). Both reactions required NADPH, NADPH-cytochrome P450 reductase, and either P450IA1 or IA2. Ketoconazole competitively inhibited both EROD and UROX activities, in microsomes from MC-treated mice. Ketoconazole also inhibited UROX catalyzed by reconstituted P450IA2. In contrast, ketoconazole did not inhibit UROX catalyzed by xanthine oxidase in the presence of iron-EDTA. Superoxide dismutase, catalase, and mannitol inhibited UROX catalyzed by xanthine oxidase/iron-EDTA, but did not affect UROX catalyzed by either microsomes or reconstituted P450IA2. These results suggest that UROX catalyzed by P450IA2 in microsomes and reconstituted systems does not involve free reactive oxygen species. Two known substrates of cytochrome P450IA2, 2-amino-3,4-dimethylimidazole[4,5-f]quinoline and phenacetin, were shown to inhibit the microsomal UROX reaction, suggesting that uroporphyrinogen binds to a substrate-binding site on the cytochrome P450. 相似文献
18.
T Watabe K Kobayashi Y Saitoh T Komatsu N Ozawa A Tsubaki K Endoh A Hiratsuka 《The Journal of biological chemistry》1986,261(7):3200-3207
Male rat liver microsomes oxidized androsta-5,16-dien-3 beta-ol (delta 16-ANDO) to delta 16-ANDO-5,6 alpha-, -5,6 beta-, -16,17 alpha-, and -16,17 beta-epoxides and delta 16-ANDO-5 alpha,6 beta-, -16 alpha,17 beta-, and -16 beta,17 alpha-glycols in the presence of an NADPH-generating system and the microsomal lipid peroxidation accelerator, Fe2+-ADP. The hepatic microsomes hydrolyzed all the delta 16-ANDO epoxides to the glycols. delta 16-ANDO-5 alpha,6 beta-glycol was the sole metabolite from both 5,6 alpha- and 5,6 beta-epoxides. Microsomal epoxide hydrolase also hydrolyzed delta 16-ANDO-16,17 alpha-epoxide specifically to the 16 beta,17 alpha-glycol and the isomeric 16,17 beta-epoxide to the 16 alpha,17 beta- and 16 beta,17 alpha-glycols approximately in the equal ratio. The delta 5-epoxidation of delta 16-ANDO by microsomes occurred only under the conditions that lipid peroxidation took place. Direct evidence was obtained for the participation of microsomal lipid hydroperoxides in the epoxidation of delta 16-ANDO by using photochemically prepared hydroperoxides of phospholipids separated from the hepatic microsomes. The hydroperoxides generated active oxygens, tentatively assigned as alk(ylper)oxy radicals, by the action of ferrous ion and epoxidized delta 16-ANDO to afford the 5,6- and 16,17-epoxides. The Fe2+-ADP-mediated epoxidation of delta 16-ANDO by the phospholipid hydroperoxides occurred preferentially at delta 5 to delta 16 and afforded the 5,6 beta-epoxide in a higher ratio than the 5,6 alpha-epoxide, similar to the Fe2+-ADP-mediated microsomal epoxidation, while the alpha-epoxide was preferentially formed to the beta-epoxide for delta 16 in the epoxidation by both systems. 相似文献
19.
Lipoxygenase plays a central role in polyunsaturated fatty acid metabolism, inaugurating the biosynthesis of eicosanoids in animals and phytooxylipins in plants. Redox cycling of the non-heme iron cofactor represents a critical element of the catalytic mechanism. Paradoxically, the isolated enzyme contains Fe(II), but the catalytically active form contains Fe(III), and the natural oxidant for the iron is the hydroperoxide product of the catalyzed reaction. Controlling the redox state of lipoxygenase iron with small molecules, inhibitors or activators, could be a means to modulate the activity of the enzyme. The effects of secondary alkyl hydroperoxides and the corresponding alcohols on soybean lipoxygenase-1 reaction rates were investigated and found to be very different. Secondary alcohols were noncompetitive or linear mixed inhibitors with inhibition constants in the millimolar concentration range, with more hydrophobic compounds producing lower values. Secondary alkyl hydroperoxides were inhibitors of lipoxygenase-1 primarily at high substrate concentration. They were more effective inhibitors than the alcohols, with dissociation constants in the micromolar concentration range. The hydroperoxides bearing longer alkyl substituents were the more effective inhibitors. Oxidation of the iron in lipoxygenase-1 by 2-hydroperoxyalkanes was evident in electron paramagnetic resonance (EPR) measurements, but the enzyme was neither activated nor was it inactivated. Instead there was evidence for an entirely different reaction catalyzed by the enzyme, a homolytic dehydration of the hydroperoxide to produce the corresponding carbonyl compound. 相似文献
20.
The mechanism of organosulfur oxygenation by peroxidases [lactoperoxidase (LPX), chloroperoxidase, thyroid peroxidase, and horseradish peroxidase] and hydrogen peroxide was investigated by use of para-substituted thiobenzamides and thioanisoles. The rate constants for thiobenzamide oxygenation by LPX/H2O2 were found to correlate with calculated vertical ionization potentials, suggesting rate-limiting single-electron transfer between LPX compound I and the organosulfur substrate. The incorporation of oxygen from 18O-labeled hydrogen peroxide, water, and molecular oxygen into sulfoxides during peroxidase-catalyzed S-oxygenation reactions was determined by LC- and GC-MS. All peroxidases tested catalyzed essentially quantitative oxygen transfer from 18O-labeled hydrogen peroxide into thiobenzamide S-oxide, suggesting that oxygen rebound from the oxoferryl heme is tightly coupled with the initial electron transfer in the active site. Experiments using H2(18)O2, 18O2, and H2(18)O showed that LPX catalyzed approximately 85, 22, and 0% 18O-incorporation into thioanisole sulfoxide oxygen, respectively. These results are consistent with a active site controlled mechanism in which the protein radical form of LPX compound I is an intermediate in LPX-mediated sulfoxidation reactions. 相似文献