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(1/145) A rat brain fraction and different purified peroxidases catalyzing the formation of dopaminochrome from dopamine.

Dopaminochrome formation is catalyzed by commercially available purified peroxidases (EC 1.11.1.7) such as horseradish, lacto- and myelo-peroxidase using dopamine, hydrogen peroxide or promethazine sulfoxide as substrates. A rat brain fraction (RBF) catalyzes a similar reaction and its catalytic power increases after preincubation with hydrogen peroxide/ascorbic acid. The activity of both the purified enzymes and the RBF preparation is inhibited by carnosine and characterized by excess substrate inhibition. The enzymes recognize different substrates but show the highest affinity for dopamine. The RBF fraction is strongly buffered against oxidation by compounds such as glutathione and by bioreductive enzymes such as DT-diaphorase (EC 1.6.99.2) which can use as a substrate menadione or dopaminochrome. The rat brain dopamine peroxidizing activity appeared to be mostly bound to the synaptosomal fraction. The reaction catalyzed by the purified peroxidases was followed by electron spin resonance spectroscopy and, unlike that catalyzed by RBF, was shown to produce the signal of a transient dopamine-o-semiquinone radical.  (+info)

(2/145) Gated and ungated electron transfer reactions from aromatic amine dehydrogenase to azurin.

Interprotein electron transfer (ET) occurs between the tryptophan tryptophylquinone (TTQ) prosthetic group of aromatic amine dehydrogenase (AADH) and copper of azurin. The ET reactions from two chemically distinct reduced forms of TTQ were studied: an O-quinol form that was generated by reduction by dithionite, and an N-quinol form that was generated by reduction by substrate. It was previously shown that on reduction by substrate, an amino group displaces a carbonyl oxygen on TTQ, and that this significantly alters the rate of its oxidation by azurin (Hyun, Y-L., and Davidson V. L. (1995) Biochemistry 34, 12249-12254). To determine the basis for this change in reactivity, comparative kinetic and thermodynamic analyses of the ET reactions from the O-quinol and N-quinol forms of TTQ in AADH to the copper of azurin were performed. The reaction of the O-quinol exhibited values of electronic coupling (H(AB)) of 0.13 cm(-1) and reorganizational energy (lambda) of 1.6 eV, and predicted an ET distance of approximately 15 A. These results are consistent with the ET event being the rate-determining step for the redox reaction. Analysis of the reaction of the N-quinol by Marcus theory yielded an H(AB) which exceeded the nonadiabatic limit and predicted a negative ET distance. These results are diagnostic of a gated ET reaction. Solvent deuterium kinetic isotope effects of 1.5 and 3.2 were obtained, respectively, for the ET reactions from O-quinol and N-quinol AADH indicating that transfer of an exchangeable proton was involved in the rate-limiting reaction step which gates ET from the N-quinol, but not the O-quinol. These results are compared with those for the ET reactions from another TTQ enzyme, methylamine dehydrogenase, to amicyanin. The mechanism by which the ET reaction of the N-quinol is gated is also related to mechanisms of other gated interprotein ET reactions.  (+info)

(3/145) Structural determinants in domain II of human glutathione transferase M2-2 govern the characteristic activities with aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine, and 1,2-dichloro-4-nitrobenzene.

Two human Mu class glutathione transferases, hGST M1-1 and hGST M2-2, with high sequence identity (84%) exhibit a 100-fold difference in activities with the substrates aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG), and 1,2-dichloro-4-nitrobenzene (DCNB), hGST M2-2 being more efficient. A sequence alignment with the rat Mu class GST M3-3, an enzyme also showing high activities with aminochrome and DCNB, demonstrated an identical structural cluster of residues 164-168 in the alpha6-helices of rGST M3-3 and hGST M2-2, a motif unique among known sequences of human, rat, and mouse Mu class GSTs. A putative electrostatic network Arg107-Asp161-Arg165-Glu164(-Gln167) was identified based on the published three-dimensional structure of hGST M2-2. Corresponding variant residues of hGSTM1-1 (Leu165, Asp164, and Arg167) as well as the active site residue Ser209 were targeted for point mutations, introducing hGST M2-2 residues to the framework of hGST M1-1, to improve the activities with substrates characteristic of hGST M2-2. In addition, chimeric enzymes composed of hGST M1-1 and hGST M2-2 sequences were analyzed. The activity with 1-chloro-2,4-dinitrobenzene (CDNB) was retained in all mutant enzymes, proving that they were catalytically competent, but none of the point mutations improved the activities with hGST M2-2 characteristic substrates. The chimeric enzymes showed that the structural determinants of these activities reside in domain II and that residue Arg165 in hGST M2-2 appears to be important for the reactions with cyanoDMNG and DCNB. A mutant, which contained all the hGST M2-2 residues of the putative electrostatic network, was still lacking one order of magnitude of the activities with the characteristic substrates of wild-type hGST M2-2. It was concluded that a limited set of point mutations is not sufficient, but that indirect secondary structural affects also contribute to the hGST M2-2 characteristic activities with aminochrome, cyanoDMNG, and DCNB.  (+info)

(4/145) Physiological importance of quinoenzymes and the O-quinone family of cofactors.

O-quinone cofactors derived from tyrosine and tryptophan are involved in novel biological reactions that range from oxidative deaminations to free-radical redox reactions. The formation of each of these cofactors appears to involve post-translational modifications of either tyrosine or tryptophan residues. The modifications result in cofactors, such as topaquinone (TPQ), tryptophan tryptophylquinone (TTQ), lysine tyrosylquinone (LTQ) or the copper-complexed cysteinyl-tyrosyl radical from metal-catalyzed reactions. Pyrroloquinoline quinone (PQQ) appears to be formed from the annulation of peptidyl glutamic acid and tyrosine residues stemming from their modification as components of a precursor peptide substrate. PQQ, a primary focus of this review, has invoked considerable interest because of its presence in foods, antioxidant properties and role as a growth-promoting factor. Although no enzymes in animals have been identified that exclusively utilize PQQ, oral supplementation of PQQ in nanomolar amounts increases the responsiveness of B- and T-cells to mitogens and improves neurologic function and reproductive outcome in rodents. Regarding TPQ and LTQ, a case may be made that the formation of TPQ and LTQ is also influenced by nutritional status, specifically dietary copper. For at least one of the amine oxidases, lysyl oxidase, enzymatic activity correlates directly with copper intake. TPQ and LTQ are generated following the incorporation of copper by a process that involves the two-step oxidation of a specified tyrosyl residue to first peptidyl dopa and then peptidyl topaquinone to generate active enzymes, generally classed as "quinoenzymes." Limited attention is also paid to TTQ and the copper-complexed cysteinyl-tyrosyl radical, cofactors important to fungal and bacterial redox processes.  (+info)

(5/145) Recombinant cytochrome P450 2D18 metabolism of dopamine and arachidonic acid.

The function of cytochrome P450 (P450) in the mammalian brain is not well understood. In an effort to further this understanding, this study identifies two endogenous substrates for P450 2D18. Previous reports have shown that this isoform is expressed in the rat brain, and the recombinant enzyme catalyzes the N-demethylation of the antidepressants imipramine and desipramine. By further examining the substrate profile of P450 2D18, inferences can be made as to potential endogenous P450 substrates. Herein we demonstrate the metabolism of the central nervous system-acting compounds chlorpromazine and chlorzoxazone with turnover numbers of 1.8 and 0. 9 nmol/min/nmol, respectively. Because the four aforementioned pharmaceutical substrates work by binding to neurotransmitter receptors, binding assays and oxidation reactions were performed to test whether dopamine is a substrate for P450 2D18. These data indicate a K(S) value of 678 microM and that P450 2D18 can support the oxidation of dopamine to aminochrome through a peroxide-shunt mechanism. We also report the P450 2D18-mediated omega-hydroxylation and epoxygenation of arachidonic acid, primarily leading to the formation of 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, compounds that have been shown to have vasoactive properties in brain, kidney, and heart tissues. The data presented herein suggest a possible role for P450 involvement in membrane and receptor regulation via epoxyeicosatrienoic acid formation and a potential involvement of P450 in the oxidation of dopamine to reactive oxygen species under aberrant physiological conditions where the sequestering of dopamine becomes compromised, such as in Parkinson's disease.  (+info)

(6/145) Nitric oxide inhibits isoproterenol-stimulated adipocyte lipolysis through oxidative inactivation of the beta-agonist.

Nitric oxide has been implicated in the inhibition of catecholamine-stimulated lipolysis in adipose tissue by as yet unknown mechanisms. In the present study, it is shown that the nitric oxide donor, 2,2-diethyl-1-nitroso-oxyhydrazine, antagonized isoproterenol (isoprenaline)-induced lipolysis in rat adipocytes, freshly isolated from white adipose tissue, by decreasing the potency of the beta-agonist without affecting its efficacy. These data suggest that nitric oxide did not act downstream of the beta-adrenoceptor but reduced the effective concentration of isoproterenol. In support of the latter hypothesis, we found that pre-treatment of isoproterenol with nitric oxide abolished the lipolytic activity of the catecholamine. Spectroscopic data and HPLC analysis confirmed that the nitric oxide-mediated inactivation of isoproterenol was in fact because of the modification of the catecholamine through a sequence of oxidation reactions, which apparently involved the generation of an aminochrome. Similarly, aminochrome was found to be the primary product of isoproterenol oxidation by 3-morpholinosydnonimine and peroxynitrite. Finally, it was shown that nitric oxide released from cytokine-stimulated adipocytes attenuated the lipolytic effect of isoproterenol by inactivating the catecholamine. In contrast with very recent findings, which suggest that nitric oxide impairs the beta-adrenergic action of isoproterenol through intracellular mechanisms and not through a chemical reaction between NO and the catecholamine, we showed that nitric oxide was able to attenuate the pharmacological activity of isoproterenol in vitro as well as in a nitric oxide-generating cellular system through oxidation of the beta-agonist. These findings should be taken into account in both the design and interpretation of studies used to investigate the role of nitric oxide as a modulator of isoproterenol-stimulated signal transduction pathways.  (+info)

(7/145) Importance of barrier shape in enzyme-catalyzed reactions. Vibrationally assisted hydrogen tunneling in tryptophan tryptophylquinone-dependent amine dehydrogenases.

C-H bond breakage by tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) occurs by vibrationally assisted tunneling (Basran, J., Sutcliffe, M. J., and Scrutton, N. S. (1999) Biochemistry 38, 3218--3222). We show here a similar mechanism in TTQ-dependent aromatic amine dehydrogenase (AADH). The rate of TTQ reduction by dopamine in AADH has a large, temperature independent kinetic isotope effect (KIE = 12.9 +/- 0.2), which is highly suggestive of vibrationally assisted tunneling. H-transfer is compromised with benzylamine as substrate and the KIE is deflated (4.8 +/- 0.2). The KIE is temperature-independent, but reaction rates are strongly dependent on temperature. With tryptamine as substrate reaction rates can be determined only at low temperature as C-H bond cleavage is rapid, and an exceptionally large KIE (54.7 +/- 1.0) is observed. Studies with deuterated tryptamine suggest vibrationally assisted tunneling is the mechanism of deuterium and, by inference, hydrogen transfer. Bond cleavage by MADH using a slow substrate (ethanolamine) occurs with an inflated KIE (14.7 +/- 0.2 at 25 degrees C). The KIE is temperature-dependent, consistent with differential tunneling of protium and deuterium. Our observations illustrate the different modes of H-transfer in MADH and AADH with fast and slow substrates and highlight the importance of barrier shape in determining reaction rate.  (+info)

(8/145) Bioactivation of tamoxifen to metabolite E quinone methide: reaction with glutathione and DNA.

Despite the beneficial effects of tamoxifen in the treatment and prevention of breast cancer, long-term usage of this popular antiestrogen has been linked to an increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. Alternatively, tamoxifen could undergo O-dealkylation to give cis/trans-1,2-diphenyl-1-(4-hydroxyphenyl)-but-1-ene, which is commonly known as metabolite E. Because of its structural similarity to 4-hydroxytamoxifen, metabolite E could also be biotransformed to a quinone methide, which has the potential to alkylate DNA and may contribute to the genotoxic effects of tamoxifen. To further probe the chemical reactivity/toxicity of such an electrophilic species, we have prepared metabolite E quinone methide chemically and enzymatically and examined its reactivity with glutathione (GSH) and DNA. Like 4-hydroxytamoxifen quinone methide, metabolite E quinone methide is quite stable; its half-life under physiological conditions is around 4 h, and its half-life in the presence of GSH is approximately 4 min. However, unlike the unstable GSH adducts of 4-hydroxytamoxifen quinone methide, metabolite E GSH adducts are stable enough to be isolated and characterized by NMR and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Reaction of metabolite E quinone methide with DNA generated exclusively deoxyguanosine adducts, which were characterized by LC/MS/MS. These data suggest that metabolite E has the potential to cause cytotoxicity/genotoxicity through the formation of a quinone methide.  (+info)