Tyrosinase catalyzes an unusual oxidative decarboxylation of 3,4-dihydroxymandelate

Tyrosinase usually catalyzes the conversion of monophenols to o-diphenols and oxidation of diphenols to the corresponding quinones. However, when 3,4-dihydroxymandelic acid was provided as the substrate, it catalyzed an unusual oxidative decarboxylation reaction generating 3,4-dihydroxybenzaldehyde as the sole product. The identity of the product was confirmed by high-performance liquid chromatography (HPLC) as well as ultraviolet and infrared spectral studies. None of the following enzymes tested catalyzed the new reaction: galactose oxidase, ceruloplasmin, superoxide dismutase, ascorbate oxidase, dopamine beta-hydroxylase, and peroxidase. Phenol oxidase inhibitors such as phenylthiourea, potassium cyanide, and sodium azide inhibited the reaction drastically, suggesting the participation of the active site copper of the enzyme in the catalysis. Mimosine, a well-known competitive inhibitor of tyrosinase, competitively inhibited the new reaction also. 4-Hydroxymandelic acid and 3-methoxy-4-hydroxymandelic acid neither served as substrates nor inhibited the reaction. Putative intermediates such as 3,4-dihydroxybenzyl alcohol and (3,4-dihydroxybenzoyl)formic acid did not accumulate during the reaction. Oxidation to a quinone methide derivative rather than conventional quinone accounts for this unusual oxidative decarboxylation reaction. Earlier from this laboratory, we reported the conversion of 4-alkylcatechols to quinone methides catalyzed by a cuticular phenol oxidase [Sugumaran, M., & Lipke, H. (1983) FEBS Lett. 155, 65-68]. Present studies demonstrate that mushroom tyrosinase will also catalyze quinone methide production with the same active site copper if a suitable substrate such as 3,4-dihydroxymandelic acid is provided.     

Biosynthesis of dehydro-N-acetyldopamine by a soluble enzyme preparation from the larval cuticle of Sarcophaga bullata involves intermediary formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide

The enzymes involved in the side chain hydroxylation and side chain desaturation of the sclerotizing precursor N-acetyldopamine (NADA) were obtained in the soluble form from the larval cuticle of Sarcophaga bullata and the mechanism of the reaction was investigated. Phenylthiourea, a well-known inhibitor of phenoloxidases, drastically inhibited both the reactions, indicating the requirement of a phenoloxidase component. N-acetylcysteine, a powerful quinone trap, trapped the transiently formed NADA quinone and prevented the production of both N-acetylnorepinephrine and dehydro NADA. Exogenously added NADA quinone was readily converted by these enzyme preparations to N-acetylnorepinephrine and dehydro NADA. 4-Alkyl-o-quinone:2-hydroxy-p-quinone methide isomerase obtained from the cuticular preparations converted chemically synthesized NADA quinone to its quinone methide. The quinone methide formed reacted rapidly and nonenzymatically with water to form N-acetylnorepinephrine as the stable product. Similarly 4-(2-hydroxyethyl)-o-benzoquinone was converted to 3,4-dihydroxyphenyl glycol. When the NADA quinone-quinone isomerase reaction was performed in buffer containing 10% methanol, beta-methoxy NADA was obtained as an additional product. Furthermore, the quinones of N-acetylnorepinephrine and 3,4-dihydroxyphenyl glycol were converted to N-acetylarterenone and 2-hydroxy-3',4'-dihydroxyacetophenone, respectively, by the enzyme. Comparison of nonenzymatic versus enzymatic transformation of NADA to N-acetylnorepinephrine revealed that the enzymatic reaction is at least 100 times faster than the nonenzymatic rate. Resolution of the NADA desaturase system on Benzamidine Sepharose and Sephacryl S-200 columns yielded the above-mentioned quinone isomerase and NADA quinone methide:dehydro NADA isomerase. The latter, on reconstitution with mushroom tyrosinase and hemolymph quinone isomerase, catalyzed the biosynthesis of dehydro NADA from NADA with the intermediary formation of NADA quinone and NADA quinone methide. The results are interpreted in terms of the quinone methide model elaborated by our group [Sugumaran: Adv. Insect Physiol. 21:179-231, 1988; Sugumaran et al.: Arch. Insect Biochem. Physiol. 11:109, 1989] and it is concluded that the two enzyme beta-sclerotization model [Andersen: Insect Biochem. 19:59-67, 375-382, 1989] is inadequate to account for various observations made on insect cuticle.     

Oxidation chemistry of 1,2-dehydro-N-acetyldopamines: direct evidence for the formation of 1,2-dehydro-N-acetyldopamine quinone

Two-electron oxidation of catecholamines either by phenol oxidase or by chemical oxidants such as sodium periodate produces their corresponding o-quinones as observable products. But, in the case of 1,2-dehydro-N-acetyldopamine, an important insect cuticular sclerotizing precursor, phenol oxidase catalyzed oxidation has been reported to generate a quinone methide analog as a transient, but first observable product. ?Sugumaran, M., Semensi, V., Kalyanaraman, B., Bruce, J. M., and Land, E. J. (1992) J. Biol. Chem. 267, 10355-10361. The corresponding quinone has escaped detection until now. However, in this paper, for the first time, we present direct evidence for the formation of dehydro-N-acetyldopamine quinone and show that it can readily be produced from the tautomeric quinone methide imine amide during the chemical oxidation of dehydro-N-acetyldopamine under acidic conditions. This situation is in sharp contrast to other known alkyl-substituted catechol oxidations, where quinone is the first observable product and quinone methide is the subsequently generated product. Dehydro-N-acetyldopamine quinone thus formed is also highly unstable. Semiempirical molecular orbital calculation also indicates that quinone methide imine amide is more stable than the quinone. Chemical considerations indicate that the quinone methide tautomer, and not the dehydro-N-acetyldopamine quinone, is responsible for crosslinking the structural proteins and chitin polymer in the insect cuticle. Therefore, the quinone methide tautomer, and not the quinone, is the key reactive intermediate aiding the hardening of insect cuticle.     

Oxidation of 3,4-dihydroxymandelic acid catalyzed by tyrosinase

Tyrosinase usually catalyzes the conversion of monophenols to o-diphenols and the oxidation of o-diphenols to the corresponding quinones. However, when 3,4-dihydroxymandelic acid was provided as the substrate, 3,4-dihydroxybenzaldehyde was produced. These results led to the proposal that tyrosinase catalyzes an unusual oxidative decarboxylation of this substrate (Sugumaran, M. (1986) Biochemistry 25, 4489-4492). However, 3,4-dihydroxybenzaldehyde is also obtained through the oxidation of 3,4-dihydroxymandelic acid by sodium periodate and on a mercury electrode. These results led to the proposal that tyrosinase catalyzes the oxidation of the substrate into o-quinone, which reacts immediately with a molecule of substrate, oxidizing it and through decarboxylation generates an intermediate (quinone methide) which transforms into 3,4-dihydroxybenzaldehyde; simultaneously, the original o-quinone is reduced to 3,4-dihydroxymandelic acid.     

Nonenzymatic transformations of enzymatically generated N-acetyldopamine quinone and isomeric dihydrocaffeiyl methyl amide quinone

We have recently demonstrated that the side chain hydroxylation of N-acetyldopamine and related compounds observed in several insects is caused by a two-enzyme system catalyzing the initial oxidation of catecholamine derivatives and subsequent isomerization of the resultant quinones to isomeric quinone methides, which undergo rapid nonenzymatic hydration to yield the observed products [Saul, S.J. and Sugumaran, M. (1989) FEBS Lett. 249, 155-158]. During our studies on o-quinone/p-quinone methide tautomerase, we observed that quinone methides are also produced nonenzymatically slowly, under physiological conditions. The quinone methide derived from N-acetyldopamine was hydrated to yield N-acetylnorepinephrine as the stable product as originally shown by Senoh and Witkop [(1959) J. Am. Chem. Soc. 81, 6222-6231], while the isomeric quinone methide from dihydrocaffeiyl methylamide exhibited a new reaction to form caffeiyl amide as the stable product. The identity of this product was established by UV and IR spectral studies and by chemical synthesis. We could not find any evidence of intramolecular cyclization of N-acetyldopamine quinone to iminochrome-type compound(s). The importance of quinone methides in these reactions is discussed.     

Insect melanogenesis. III. Metabolon formation in the melanogenic pathway-regulation of phenoloxidase activityy by endogenous dopachrome isomerase (decarboxylating) from Manduca sexta

Tyrosinase initiates melanogenesis in a variety of organisms. The nature of melanin formed is modified subsequently by dopachrome isomerase and other melanogenic proteins. Earlier, we reported the partial purification of dopachrome isomerase (decarboxylating) from the hemolymph of Manduca sexta and demonstrated the generation of a new quinone methide intermediate during melanogenesis (Sugumaran, M., and Semensi, V. (1991) J. Biol. Chem. 266, 6073-6078). In this paper, we report the purification of this enzyme to homogeneity and a novel inhibition mechanism for regulation of phenoloxidase activity. The activity of phenoloxidase isolated from M. sexta was markedly inhibited by purified dopachrome isomerase. In turn, phenoloxidase also reciprocated by inhibiting the isomerase activity. Preformed dopaminechrome did not serve as the substrate for the isomerase; but dopaminechrome that generated in situ by phenoloxidase was readily converted to melanin pigment by the phenoloxidase/isomerase mixture. Furthermore, the isomerase, which has a molecular weight of about 40,000 in native state, exhibited retardation during affinity electrophoresis on sodium dodeyl sulfate (SDS)-polyacrylamide gel electrophoresis gel copolymerized with tyrosinase and migrated with a molecular weight of 50,000, indicating complex formation with phenoloxidase. Electrophoresis of pupal cuticular extract on polyacrylamide gel, followed by activity staining revealed the presence of a protein band carrying both phenoloxidase and isomerase activity. Accordingly, a high-molecular-weight melanogenic complex was isolated from the pharate cuticle of M. sexta. The complex catalyzed the generation of melanochrome from dopa, while the free phenoloxidase produced only dopachrome from the same substrate. When the complex was treated with trace amounts of SDS, which inhibited the activity of dopachrome isomerase present in the complex, then only the conversion of dopa to dopachrome was observed. These studies confirm the formation of a melanogenic complex between phenoloxidase and dopachrome isomerase. By forming a complex and regulating each other's activity, these two enzymes seem to control the levels of endogenous quinones.     

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.     

Chemical and enzymic oxidation by tyrosinase of 3,4-dihydroxymandelate.

Tyrosinase usually catalyses the conversion of monophenols into o-diphenols and the oxidation of diphenols to the corresponding o-quinones. Sugumaran [(1986) Biochemistry 25, 4489-4492] has previously proposed an unusual oxidative decarboxylation of 3,4-dihydroxymandelate catalysed by tyrosinase. Our determination of the intermediates involved in the reaction demonstrated that 3,4-dihydroxybenzaldehyde is not the first intermediate appearing in the medium during the enzymic reaction. Re-examination of this new activity of tyrosinase has demonstrated that the product of the enzyme action is the o-quinone, which, owing to its instability, evolves to the final product, 3,4-dihydroxybenzaldehyde, by a chemical reaction of oxidative decarboxylation.     

Autoxidative and cyclooxygenase-2 catalyzed transformation of the dietary chemopreventive agent curcumin

The efficacy of the diphenol curcumin as a cancer chemopreventive agent is limited by its chemical and metabolic instability. Non-enzymatic degradation has been described to yield vanillin, ferulic acid, and feruloylmethane through cleavage of the heptadienone chain connecting the phenolic rings. Here we provide evidence for an alternative mechanism, resulting in autoxidative cyclization of the heptadienone moiety as a major pathway of degradation. Autoxidative transformation of curcumin was pH-dependent with the highest rate at pH 8 (2.2 μM/min) and associated with stoichiometric uptake of O(2). Oxidation was also catalyzed by recombinant cyclooxygenase-2 (COX-2) (50 nm; 7.5 μM/min), and the rate was increased ≈10-fold by the addition of 300 μM H(2)O(2). The COX-2 catalyzed transformation was inhibited by acetaminophen but not indomethacin, suggesting catalysis occurred by the peroxidase activity. We propose a mechanism of enzymatic or autoxidative hydrogen abstraction from a phenolic hydroxyl to give a quinone methide and a delocalized radical in the heptadienone chain that undergoes 5-exo cyclization and oxygenation. Hydration of the quinone methide (measured by the incorporation of O-18 from H(2)(18)O) and rearrangement under loss of water gives the final dioxygenated bicyclopentadione product. When curcumin was added to RAW264.7 cells, the bicyclopentadione was increased 1.8-fold in cells activated by LPS; vanillin and other putative cleavage products were negligible. Oxidation to a reactive quinone methide is the mechanistic basis of many phenolic anti-cancer drugs. It is possible, therefore, that oxidative transformation of curcumin, a prominent but previously unrecognized reaction, contributes to its cancer chemopreventive activity.     

Mechanism of activation of 1,2-dehydro-N-acetyldopamine for cuticular sclerotization

The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in β-sclerotization. While Andersen has indicated that dehydro NADA quinone is the β-sclerotizing agent [Andersen, 1989], we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 1988]. Since dehydro NADA quinone has not been isolated or identified till to date, we studied the enzymatic oxidation of dehydro NADA in the presence of quinone traps to characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trapped the transiently formed dehydro NADA quinone as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred. The structure of the adducts is in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with the fact that 4-hydroxyl or amino-substituted quinones instantly transformed into p-quinonoid structure, indicates that dehydro NADA quinone is only a transient intermediate and that it is the dehydro NADA quinone methide that is the thermodynamically stable product. However, since this compound is chemically more reactive due to the presence of both quinone methide and acylimine structure on it, the two side chain carbon atoms are “activated.” Based on these considerations, it is suggested that the quinone methide derived from dehydro NADA is the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during β-sclerotization.     

Role of mono- and divalent metal cations in the catalysis by yeast aldolase

The rate of deuterium exchange between [1-(S)-2H]dihydroxyacetone 3-phosphate and the solvent catalyzed by native and metal-substituted yeast aldolases has been measured. In the presence of 0.1 M potassium acetate at 15 degrees C, pH 7.3, the deuterium exchange reaction catalyzed by native yeast aldolase has a kcat of 95 s-1. In contrast to the 7-fold activity enhancement by 0.1 M potassium ion (relative to 0.1 M sodium ion) of the cleavage of D-fructose 1,6-bisphosphate catalyzed by native yeast aldolase, a negligible (1.1-fold) activation by 0.1 M potassium ion is observed in the rate of dedeuteration of [1(S)-2H]dihydroxyacetone 3-phosphate. The order of reactivity of the yeast metalloaldolases in the deuterium exchange roughly parallels that seen in the fructose bisphosphate cleavage reaction. These findings suggest that the carbonyl groups of enzyme-bound D-fructose 1,6-bisphosphate and dihydroxyacetone phosphate are both polarized by the active site divalent metal cation. A mechanistic formulation consistent with the results of this and the previous paper is presented.     

The toxicity of menadione (2-methyl-1,4-naphthoquinone) and two thioether conjugates studied with isolated renal epithelial cells

Menadione (2-methyl-1,4-naphthoquinone) was used as a model compound to test the hypothesis that thioether conjugates of quinones can be toxic to tissues associated with their elimination through a mechanism involving oxidative stress. Unlike menadione, the glutathione (2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone; MGNQ) and N-acetyl-L-cysteine (2-methyl-3-(N-acetylcysteine-S-yl)-1,4-naphthoquinone; M(NAC)NQ) thioether conjugates were not able to arylate protein thiols but were still able to redox cycle with cytochrome c reductase/NADH and rat kidney microsomes and mitochondria. Interestingly, menadione and M(NAC)NQ were equally toxic to isolated rat renal epithelial cells (IREC) while MGNQ was nontoxic. The toxicity of both menadione and M(NAC)NQ was preceded by a rapid depletion of soluble thiols and was associated with a depletion of soluble thiols and was associated with a depletion of protein thiols. Treatment of IREC with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1-nitrosourea, potentiated the thiol depletion and toxicity observed with menadione and M(NAC)NQ indicating the involvement of oxidative stress in this model of renal cell toxicity. The lack of MGNQ toxicity can be attributed to an intramolecular cyclization reaction which destroys the quinone nucleus and therefore eliminates its ability to redox cycle. These findings have important implications with regard to our understanding of the toxic potential of quinone thioether conjugates and of quinone toxicity in general.     

Reaction of the molybdenum- and copper-containing carbon monoxide dehydrogenase from Oligotropha carboxydovorans with quinones

Carbon monoxide dehydrogenase (CODH) from Oligotropha carboxydovorans catalyzes the oxidation of carbon monoxide to carbon dioxide, providing the organism both a carbon source and energy for growth. In the oxidative half of the catalytic cycle, electrons gained from CO are ultimately passed to the electron transport chain of the Gram-negative organism, but the proximal acceptor of reducing equivalents from the enzyme has not been established. Here we investigate the reaction of the reduced enzyme with various quinones and find them to be catalytically competent. Benzoquinone has a k(ox) of 125.1 s(-1) and a K(d) of 48 μM. Ubiquinone-1 has a k(ox)/K(d) value of 2.88 × 10(5) M(-1) s(-1). 1,4-Naphthoquinone has a k(ox) of 38 s(-1) and a K(d) of 140 μM. 1,2-Naphthoquinone-4-sulfonic acid has a k(ox)/K(d) of 1.31 × 10(5) M(-1) s(-1). An extensive effort to identify a cytochrome that could be reduced by CO/CODH was unsuccessful. Steady-state studies with benzoquinone indicate that the rate-limiting step is in the reductive half of the reaction (that is, the reaction of oxidized enzyme with CO). On the basis of the inhibition of CODH by diphenyliodonium chloride, we conclude that quinone substrates interact with CODH at the enzyme's flavin site. Our results strongly suggest that CODH donates reducing equivalents directly to the quinone pool without using a cytochrome as an intermediary.     

Vitamin K, 2,3-Epoxide And Quinone Reduction: Mechanism And Inhibition

The chemical and enzymatic pathways of vitamin K₁ epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase. E.C. I.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes. but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K₁ epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K, 10^?5 as rapidly as it reduced dichlorophenylindophenol(DCPIP). Reduction of 10 μM vitamin K, by200 μM NADH was not inhibited by 10μM dicoumarol. whereas DCPIP reduction was fully inhibited. In contrast to vitamin K, (menadione). vitamin K₁ (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions. suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K₁ epoxide and quinone. and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin. lapachol. and sulphaquinoxaline     

On the oxidation of 3,4-dihydroxyphenethyl alcohol and 3,4-dihydroxyphenyl glycol by cuticular enzyme(s) from Sarcophaga bullata

The catabolic fate of 3,4-dihydroxyphenethyl alcohol (DHPA) and 3,4-dihydroxyphenylethyl glycol (DHPG) in insect cuticle was determined for the first time using cuticular enzyme(s) from Sarcophaga bullata and compared with mushroom tyrosinase-medicated oxidation. Mushroom tyrosinase converted both DHPA and DHPG to their corresponding quinone derivatives, while cuticular enzyme(s) partly converted DHPA to DHPG. Cuticular enzyme(s)-mediated oxidation of DHPA also accompanied the covalent binding of DHPA to the cuticle. Cuticle-DHPA adducts, upon pronase digestion, released peptides that had bound catechols. 3,4-Dihydroxyphenyl-acetaldehyde, the expected product of side chain desaturation of DHPA, was not formed at all. The presence of N-acetylcysteine, a quinone trap, in the reaction mixture containing DHPA and cuticle resulted in the generation of DHPA-quinone-N-acetylcysteine adduct and total inhibition of DHPG formation. The insect enzyme(s) converted DHPG to its quinone at high substrate concentration and to 2-hydroxy-3′,4′-dihydroxyacetophenone at low concentration. They converted exogenously added DHPA-quinone to DHPG, but acted sluggishly on DHPG-quinone. These results are consistent with the enzymatic transformations of phenoloxidase-generated quinones to quinone methides and subsequent nonenzymatic transformation of the latter to the observed products. Thus, quinone methide formation in insect cuticle seems to be caused by the combined action of two enzymes, phenoloxidase and quinone tautomerase, rather than the action of quinone methide-generating phenoloxidase (Sugumaran: Arch Insect Biochem Physiol 8, 73–88, 1988). It is proposed that DHPA and DHPG in combination can be used effectively to examine the participation of (1) quinone, (2) quinone methide, and (3) dehydro derivative intermediates in the metabolism of 4-alkylcatechols for cuticular sclerotization.     

Dicoumarol-sensitive glucuronidation of benzo(a)pyrene metabolites in rat liver microsomes

The effect of dicoumarol on glucuronidation of 3-OH-benzo(a)pyrene (BP) appears to be due to inhibition of UDPglucuronosyltransferase (UDPGT) and not to an inhibited DT-diaphorase (NAD(P)H:quinone oxidoreductase); to date the only enzyme known to be inhibited by dicoumarol. This dicoumarol-sensitive form of UDPGT does not seem to be identical to the major form catalyzing the glucuronidation of p-nitrophenol or methylumbelliferone, nor to the isozyme involved in the formation of phenolphthalein glucuronides. These conclusions are based on the following observations: In solubilized microsomes, devoid of DT-diaphorase, a 3-OH-BP glucuronidation activity is found which is very similar to that observed in microsomes before passing through an azodicoumarol Sepharose 6B column that binds more than 98% of DT-diaphorase; in the eluate from this column the inhibition by dicoumarol of 3-OH-BP glucuronidation is the same as in microsomes containing DT-diaphorase; other coumarin derivatives, which are either modified or substituted in the methylene bridge between the two coumarin entities in dicoumarol, are potent inhibitors of DT-diaphorase but not of UDPGT; a concentration of 10(-6) M dicoumarol is sufficient to inhibit 3-OH-BP glucuronidation 50%. In contrast, to inhibit glucuronidation of p-nitrophenol or methylumbelliferone the concentration of dicoumarol must be raised to the substrate level: i.e., 10(-4) M. Phenolphthalein glucuronidation is almost unaffected even by this high concentration of dicoumarol. The present investigation also reveals that DT-diaphorase and NADPH-cytochrome P-450 reductase can both catalyze the reduction of BP-3,6-quinone for the formation of BP-3,6-quinol glucuronides. In the eluate from the azodicoumarol Sepharose 6B column, no NADH-supported glucuronidation of BP-3,6-quinone can be detected unless DT-diaphorase is added. However, NADPH-supported formation of BP-3,6-quinol glucuronides can still be observed. The rate of the latter reaction is sufficient enough to allow studies on the effect of dicoumarol on BP-3,6-quinone glucuronidation. These results show that glucuronidation of BP-3,6-quinols is also catalyzed by a dicoumarol-sensitive UDPGT. However, not only is the formation of BP-3,6-quinol monoglucuronides inhibited by dicoumarol, but the conversion of monoglucuronides to diglucuronides is inhibited as well. The former reaction is inhibited 50% by 3.5 X 10(-6) M dicoumarol (close to the I50 for 3-OH-BP glucuronidation), whereas 10 times less dicoumarol (2 X 10(-7) M) is sufficient for 50% inhibition of the latter reaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spectroscopic observation of intermediates formed during the oxidative half-reaction of copper/topa quinone-containing phenylethylamine oxidase.

The catalytic reaction of copper/topa quinone (TPQ) containing amine oxidase consists of the initial, well-characterized, reductive half-reaction and the following, less studied, oxidative half-reaction. We have analyzed the oxidative half-reaction catalyzed by phenylethylamine oxidase from Arthrobacter globiformis (AGAO) by rapid-scan stopped-flow measurements. Upon addition of dioxygen to the substrate-reduced AGAO at pH 8.2, the absorption bands derived from the semiquinone (TPQ(sq)) and aminoresorcinol forms of the TPQ cofactor disappeared within the dead time (<1 ms) of the measurements, indicating that the reaction of the substrate-reduced enzyme with dioxygen is very rapid. Concomitantly, an early intermediate exhibiting an absorption band at about 410 nm was formed, which then decayed with a rate constant of 390 +/- 50 s(-1). This intermediate was detected more prominently in the reaction in D2O buffer (pD 8.1) and was assigned to a Cu(II)-peroxy species. The assignment was based on the observation that addition of H2O2 to the substrate-reduced AGAO under anaerobic conditions led to the formation of a new band at about 415 nm, accompanied by partial quenching of absorption bands derived from TPQ(sq). Other intermediates exhibiting absorption bands at about 310 and 340 nm were also observed in the oxidative half-reaction. Kinetics of the disappearance of these latter bands did not correspond with that of the Cu(II)-peroxy band at 410 nm but did well with that of the increase of the 480 nm absorption band due to the reoxidized TPQ. Rapid increase of the absorption in the 320-370 nm region was also observed for the reaction of the substrate-reduced, Ni-substituted enzyme with dioxygen. On the basis of these results, a possible mechanism is proposed for the oxidative half-reaction of the bacterial copper amine oxidase.     

New pterocarpanquinones: Synthesis,antineoplasic activity on cultured human malignant cell lines and TNF-α modulation in human PBMC cells

A new pterocarpanquinone (5a) was synthesized through a palladium catalyzed oxyarylation reaction and was transformed, through electrophilic substitution reaction, into derivatives 5b–d. These compounds showed to be active against human leukemic cell lines and human lung cancer cell lines. Even multidrug resistant cells were sensitive to 5a, which presented low toxicity toward peripheral blood mononuclear cells (PBMC) cells and decreased the production of TNF-α by these cells. In the laboratory these pterocarpanquinones were reduced by sodium dithionite in the presence of thiophenol at physiological pH, as NAD(P)H quinone oxidoredutase-1 (NQO1) catalyzed two-electron reduction, and the resulting hydroquinone undergo structural rearrangements, leading to the formation of Michael acceptors, which were intercepted as adducts of thiophenol. These results suggest that these compounds could be activated by bioreduction.     

An NADH:quinone oxidoreductase of the halotolerant bacterium Ba1 is specifically dependent on sodium ions

The rate of NADH oxidation by inverted membrane vesicles prepared from the halotolerant bacterium Ba1 of the Dead Sea is increased specifically by sodium ions, as observed earlier in whole cells. The site of this sodium effect is identified as the NADH: quinone oxidoreductase, similarly to the other such system known, Vibrio alginolyticus (H. Tokuda and T. Unemoto (1984) J. Biol. Chem. 259, 7785-7790). Sodium accelerates quinone reduction severalfold, but oxidation of the quinol, with oxygen as terminal electron acceptor, is unaffected. The sodium-dependent pathway of quinone reduction exhibits higher apparent affinity to extraneous quinone (Q-2) than the sodium-insensitive pathway, and is specifically inhibited by 2-heptyl-4-hydroxyquinoline N-oxide. ESR spectra of the membranes contain a feature at g = 1.98 which is tentatively identified as one originating from semiquinone. This feature is increased by NADH and decreased by addition of Na+, suggesting that, as proposed from different kinds of evidence for the V. alginolyticus system, sodium affects the semiquinone reduction step. As in the other system, the site of sodium stimulation in Ba1 probably corresponds to the site of sodium translocation, which was shown earlier (S. Ken-Dror, R. Shnaiderman, and Y. Avi-Dor (1984) Arch. Biochem. Biophys. 229, 640-649) to be linked directly to a redox reaction in the respiratory chain.     

The NADPH:quinone oxidoreductase P1-zeta-crystallin in Arabidopsis catalyzes the alpha,beta-hydrogenation of 2-alkenals: detoxication of the lipid peroxide-derived reactive aldehydes

P1-zeta-crystallin (P1-ZCr) is an oxidative stress-induced NADPH:quinone oxidoreductase in Arabidopsis thaliana, but its physiological electron acceptors have not been identified. We found that recombinant P1-ZCr catalyzed the reduction of 2-alkenals of carbon chain C(3)-C(9) with NADPH. Among these 2-alkenals, the highest specificity was observed for 4-hydroxy-(2E)-nonenal (HNE), one of the major toxic products generated from lipid peroxides. (3Z)-Hexenal and aldehydes without alpha,beta-unsaturated bonds did not serve as electron acceptors. In the 2-alkenal molecules, P1-ZCr catalyzed the hydrogenation of alpha,beta-unsaturated bonds, but not the reduction of the aldehyde moiety, to produce saturated aldehydes, as determined by gas chromatography/mass spectrometry. We propose the enzyme name NADPH:2-alkenal alpha,beta-hydrogenase (ALH). A major portion of the NADPH-dependent HNE-reducing activity in A. thaliana leaves was inhibited by the specific antiserum against P1-ZCr, indicating that the endogenous P1-ZCr protein has ALH activity. Because expression of the P1-ZCr gene in A. thaliana is induced by oxidative stress treatments, we conclude that P1-ZCr functions as a defense against oxidative stress by scavenging the highly toxic, lipid peroxide-derived alpha,beta-unsaturated aldehydes.