Inactivation of Tryptophan Hydroxylase by Nitric Oxide: Enhancement by Tetrahydrobiopterin

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by the nitric oxide generators sodium nitroprusside, diethylamine/nitric oxide complex, and S -nitroso- N -acetylpenicillamine. Physiological concentrations of tetrahydrobiopterin, the natural and endogenous cofactor for the hydroxylase, significantly enhance the inactivation of the enzyme caused by each of these nitric oxide generators. The substrate tryptophan does not have this effect. The chemically reduced (tetrahydro-) form of the pterin is required for the enhancement, because neither biopterin nor dihydrobiopterin is effective. The 6 S -isomer of tetrahydrobiopterin, which has little cofactor efficacy for tryptophan hydroxylase, does not enhance enzyme inactivation as does the natural 6 R -isomer. A number of synthetic, reduced pterins share with tetrahydrobiopterin the ability to enhance nitric oxide-induced inactivation of tryptophan hydroxylase. The tetrahydrobiopterin effect is not prevented by agents known to scavenge hydrogen peroxide, superoxide radicals, peroxynitrite anions, hydroxyl radicals, or singlet oxygen. On the other hand, cysteine partially protects the enzyme from both the nitric oxide-induced inactivation and the combined pterin/nitric oxide-induced inactivation. These results suggest that the tetrahydrobiopterin cofactor enhances the nitric oxide-induced inactivation of tryptophan hydroxylase via a mechanism that involves attack on free protein sulfhydryls. Potential in vivo correlates of a tetrahydrobiopterin participation in the inactivation of tryptophan hydroxylase can be drawn to the neurotoxic amphetamines.     

Interaction of rhodanese with intermediates of oxygen reduction

Cyanide-promoted inactivation of the enzyme rhodanese [thiosulfate sulfurtransferase (EC 2.8.1.1)] in the presence of ketoaldehydes is caused by reduced forms of molecular oxygen generated during autoxidation of the reaction products. The requirement of both catalase and superoxide dismutase to prevent rhodanese inactivation indicates that hydroxyl radical could be the most efficient inactivating agent. Rhodanese, also in the less stable sulfur-free form, shows a different sensitivity towards oxygen activated species. While the enzyme is unaffected by superoxide radical, it is rapidly inactivated by hydrogen peroxide. The extent of inactivation depends on the molar ratio between sulfur-free enzyme and oxidizing agent. Fully inactive enzyme is reactivated by reduction with its substrate thiosulfate.     

Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.     

A permissive apoptotic environment: function of a decrease in intracellular superoxide anion and cytosolic acidification.

Reactive oxygen species are involved in cellular processes as diverse as proliferation and cell death. At concentrations that do not overwhelm the cellular antioxidant defense systems, reactive oxygen species such as superoxide anion can inhibit death signaling. The sensitivity of cells to apoptotic triggers is significantly increased upon decreasing intracellular superoxide concentration. The critical determinant is the tight intracellular balance between superoxide and hydrogen peroxide levels, and a shift from the tightly regulated physiological ratio could impact cellular response to death stimuli. A shift toward hydrogen peroxide leads to activation of the effector components of the cells' apoptotic machinery by inducing reduction of the intracellular milieu and a drop in cytosolic pH, thereby creating a facilitative environment for efficient death execution. Hence, we propose that a permissive apoptotic milieu is a function of decreased intracellular superoxide concentration and cytosolic acidification.     

Vibrio cholerae periplasmic superoxide dismutase: isolation of the gene and overexpression of the protein

Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species. Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase. Moreover, we have set up an expression system yielding large amounts of V. cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form. Unlike the bovine enzyme, V. cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide. This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.     

Oxidase Reaction of the Hybrid Mn-Peroxidase of the Fungus <Emphasis Type="Italic">Panus tigrinus</Emphasis> 8/18

The hybrid Mn-peroxidase of the fungus Panus tigrinus 8/18 oxidized NADH in the absence of hydrogen peroxide, this being accompanied by the consumption of oxygen. The reaction of NADH oxidation started after a period of induction and completely depended on the presence of Mn(II). The reaction was inhibited in the presence of catalase and super-oxide dismutase. Oxidation of NADH by the enzyme or by manganese(III)acetate was accompanied by the production of hydrogen peroxide and superoxide radicals. In the presence of NADH, the enzyme was transformed into a catalytically inactive oxidized form (compound III), and the latter was inactivated with bleaching of the heme. The substrate of the hybrid Mn-peroxidase (Mn(II)) reduced compound III to yield the native form of the enzyme and prevented its inactivation. It is assumed that the hybrid Mn-peroxidase used the formed hydrogen peroxide in the usual peroxidase reaction to produce Mn(III), which was involved in the formation of hydrogen peroxide and thus accelerated the peroxidase reaction. The reaction of NADH oxidation is a peroxidase reaction and the consumption of oxygen is due to its interaction with the products of NADH oxidation. The role of Mn(II) in the oxidation of NADH consisted in the production of hydrogen peroxide and the protection of the enzyme from inactivation.__________Translated from Biokhimiya, Vol. 70, No. 4, 2005, pp. 568–574.Original Russian Text Copyright © 2005 by Lisov, Leontievsky, Golovleva.     

Superoxide anion: Oncogenic reactive oxygen species?

Recent evidence linking intracellular reactive oxygen species to cell survival and/or proliferation signals has resulted in a paradigm shift from the age-old dogma implicating reactive oxygen species exclusively in cell damage and death. It is now accepted that reactive oxygen species play important roles in normal physiological states and that depending on the species involved the effect could be highly varied. In this regard, the effects of the two major reactive oxygen species, superoxide and hydrogen peroxide have been extensively studied. During normal cell growth a tight balance between the two species is kept under check by the cells' anti-oxidant defense systems. Deficiency or defect in this defense armory is invariably associated with neoplasia, thus rendering the intracellular redox status in a state of imbalance and generating a "pro-oxidant" milieu. A variety of model systems have underscored the relationship between a pro-oxidant state and cancer promotion and progression. In this review, we present evidence to support the hypothesis that the effect of intracellular reactive oxygen species on oncogenesis is dependent on the ratio of intracellular superoxide to hydrogen peroxide in that a predominant increase in superoxide supports cell survival and promotes oncogenesis whereas a tilt in favor of hydrogen peroxide prevents carcinogenesis by facilitating cell death signaling.     

Reducing sugars can induce the oxidative inactivation of rhodanese.

The enzyme rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) is inactivated on incubation with reducing sugars such as glucose, mannose, or fructose, but is stable with non-reducing sugars or related polyhydroxy compounds. The enzyme is inactivated with (ES) or without (E) the transferable sulfur atom, although E is considerably more sensitive, and inactivation is accentuated by cyanide. Inactivation of E is accompanied by increased proteolytic susceptibility, a decreased sulfhydryl titer, a red-shift and quenching of the protein fluorescence, and the appearance of hydrophobic surfaces. Superoxide dismutase and/or catalase protect rhodanese. Inactive enzyme can be partially reactivated during assay and almost completely reactivated by incubation with thiosulfate, lauryl maltoside, and 2-mercaptoethanol. These results are similar to those observed when rhodanese is inactivated by hydrogen peroxide. These observations, as well as the cyanide-dependent, oxidative inactivation by phenylglyoxal, are explained by invoking the formation of reactive oxygen species such as superoxide or hydrogen peroxide from autooxidation of alpha-hydroxy carbonyl compounds, which can be facilitated by cyanide.     

Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay)

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.     

Oxidative metabolism of human platelets.

The reducing capacity toward cytochrome c present in human resting platelets increases upon platelet stimulation, and is partially inhibited by superoxide dismutase. This activity therefore represents the generation of superoxide anion. In order to evaluate hydrogen peroxide formation a quantitative assay by mean of dichlorofluorescin (DCFH) has been set up. The DCFH, trapped inside the cell, is oxidized by hydrogen peroxide to the fluorescent compound DCF. Basal DCF increases during activation of platelets by agonists. Arachidonic acid, calcium ionophore A23187 and to a lesser extent PMA and thrombin are the most effective. N-ethylmaleimide induces a dose-dependent DCFH oxidation and potentiates the effect of agonists. NAD(P)H--cytochrome c reductase enzyme, which catalyzes superoxide anion production, is present in platelets at high specific activity, as well as those enzymes who protect the cells from oxygen reactive species.     

Damage to the oxygen-evolving complex by superoxide anion, hydrogen peroxide, and hydroxyl radical in photoinhibition of photosystem II

Under strong illumination of a photosystem II (PSII) membrane, endogenous superoxide anion, hydrogen peroxide, and hydroxyl radical were successively produced. These compounds then cooperatively resulted in a release of manganese from the oxygen-evolving complex (OEC) and an inhibition of oxygen evolution activity. The OEC inactivation was initiated by an acceptor-side generated superoxide anion, and hydrogen peroxide was most probably responsible for the transportation of reactive oxygen species (ROS) across the PSII membrane from the acceptor-side to the donor-side. Besides ROS being generated in the acceptor-side induced manganese loss; there may also be a ROS-independent manganese loss in the OEC of PSII. Both superoxide anion and hydroxyl radical located inside the PSII membrane were directly identified by a spin trapping-electron spin resonance (ESR) method in combination with a lipophilic spin trap, 5-(diethoxyphosphoryl)-5-phenethyl-1-pyrroline N-oxide (DEPPEPO). The endogenous hydrogen peroxide production was examined by oxidation of thiobenzamide.     

Leukocyte-mediated inactivation of alpha 1-proteinase inhibitor is inhibited by amino analogues of alpha-tocopherol.

Human leukocytes stimulated by opsonized zymosan increase their NADPH oxidase-catalysed reduction of molecular oxygen. This leads to enhanced formation of superoxyl radicals and subsequently hydrogen peroxide. The leukocyte enzyme myeloperoxidase generates the strong microbicidal oxidant hypochlorite from hydrogen peroxide and chloride anions. Hypochlorite inactivates serum alpha 1-proteinase inhibitor, a protein which protects host tissue from digestion by proteinases, that are also secreted by stimulated leukocytes. Micromolar concentrations of a water-soluble, quaternary ammonium analogue of alpha-tocopherol (vitamin E) (3,4-dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopyran-2 -ethanaminium 4-methylbenzenesulfonate) and its tertiary amine derivative (3,4-dihydro-2- (2-dimethylaminoethyl)-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol hydrochloride) were able to protect alpha 1-proteinase inhibitor from inactivation by stimulated human leukocytes. The mechanism of action of the quaternary ammonium analogue was further investigated. Selective inhibition of hydrogen peroxide formation is assumed to be the reason for its protective effect. This compound rapidly reacts with superoxyl radicals, but not with hydrogen peroxide, and is only a weak hypochlorite scavenger. It neither impedes exocytosis of elastase, nor effectively inhibits NADPH oxidase or myeloperoxidase. In contrast, superoxide dismutase, which enhances hydrogen peroxide formation, cannot protect alpha 1-proteinase inhibitor from inactivation.     

Effect of reactive oxygen species on the metabolism of tryptophan in rat brain: Influence of age

In an earlier study, oxidation of tryptophan hydroxylase was implicated as its affinity was decreased with aging in rat brain. To establish any potential link between its oxidative damage and aging, we have determined the activities of antioxidant enzymes in midbrain, pons and medulla of 2, 12 and 24 month old Fisher 344 BNF1 rats. The results obtained suggest that the activities of antioxidant enzymes varied considerably with age and brain regions studied. Activities of Cu/Zn superoxide dismutase and glutathione peroxidase were found to increase from 2 to 12 months and then decrease in 24 month old rats. However catalase activity decreased consistently with the age. A parallel increase in the carbonyl content was observed in these brain regions indicating the oxidation of proteins. Reactive oxygen species when included in the incubation mixture decreased the activity of tryptophan hydroxylase in a concentration dependent manner. The loss of tryptophan hydroxylase activity induced by hydrogen peroxide and superoxide anion was prevented by catalase. However superoxide dismutase did not provide such protection. Sulfhydryl agents, cysteine, glutathione and dithiothreitol partially prevented the loss of activity. These studies suggest an involvement of reactive oxygen species for sulfhydryl oxidation of tryptophan hydroxylase in aging.     

Human SOD2 modification by dopamine quinones affects enzymatic activity by promoting its aggregation: possible implications for Parkinson's disease

Mitochondrial dysfunction and oxidative stress are considered central in dopaminergic neurodegeneration in Parkinson's disease (PD). Oxidative stress occurs when the endogenous antioxidant systems are overcome by the generation of reactive oxygen species (ROS). A plausible source of oxidative stress, which could account for the selective degeneration of dopaminergic neurons, is the redox chemistry of dopamine (DA) and leads to the formation of ROS and reactive dopamine-quinones (DAQs). Superoxide dismutase 2 (SOD2) is a mitochondrial enzyme that converts superoxide radicals to molecular oxygen and hydrogen peroxide, providing a first line of defense against ROS. We investigated the possible interplay between DA and SOD2 in the pathogenesis of PD using enzymatic essays, site-specific mutagenesis, and optical and high-field-cw-EPR spectroscopies. Using radioactive DA, we demonstrated that SOD2 is a target of DAQs. Exposure to micromolar DAQ concentrations induces a loss of up to 50% of SOD2 enzymatic activity in a dose-dependent manner, which is correlated to the concomitant formation of protein aggregates, while the coordination geometry of the active site appears unaffected by DAQ modifications. Our findings support a model in which DAQ-mediated SOD2 inactivation increases mitochondrial ROS production, suggesting a link between oxidative stress and mitochondrial dysfunction.     

Pteridine-dependent oxygen activation in neutrophils

We investigated the influence of neopterin and 7,8-dihydroneopterin on the myeloperoxidase activity and secretory degranulation in neutrophils and interaction of pteridines with its major substrate (hydrogen peroxide) and intermediate product of halogenation cycle (hypochlorous acid). It was shown that, in neutrophils, the redox-pair, neopterin and 7,8-dihydroneopterin, control oxygen activation, which is regulated by myeloperoxidase. Pteridines influence the secretion of myeloperoxidase depending on concentration and decrease the level of hydrogen peroxide and hypochlorous acid, which are the substrate and intermediate product of the enzyme, respectively. It was found that, in micromolar concentrations, 7,8-dihydroneopterin is a noncompetitive inhibitor of myeloperoxidase. We suppose that myeloperoxidase facilitates 7,8-dihydroneopterin oxidation by hypochlorous acid and results in an increase in neopterin concentration. These changes modify the concentration of intracellular and extracellular reactive oxygen species.     

Structural Analysis of Peroxide-Soaked MnSOD Crystals Reveals Side-On Binding of Peroxide to Active-Site Manganese

The superoxide dismutase (SOD) enzymes are important antioxidant agents that protect cells from reactive oxygen species. The SOD family is responsible for catalyzing the disproportionation of superoxide radical to oxygen and hydrogen peroxide. Manganese- and iron-containing SOD exhibit product inhibition whereas Cu/ZnSOD does not. Here, we report the crystal structure of Escherichia coli MnSOD with hydrogen peroxide cryotrapped in the active site. Crystallographic refinement to 1.55 Å and close inspection revealed electron density for hydrogen peroxide in three of the four active sites in the asymmetric unit. The hydrogen peroxide molecules are in the position opposite His26 that is normally assumed by water in the trigonal bipyramidal resting state of the enzyme. Hydrogen peroxide is present in active sites B, C, and D and is side-on coordinated to the active-site manganese. In chains B and D, the peroxide is oriented in the plane formed by manganese and ligands Asp167 and His26. In chain C, the peroxide is bound, making a 70° angle to the plane. Comparison of the peroxide-bound active site with the hydroxide-bound octahedral form shows a shifting of residue Tyr34 towards the active site when peroxide is bound. Comparison with peroxide-soaked Cu/ZnSOD indicates end-on binding of peroxide when the SOD does not exhibit inhibition by peroxide and side-on binding of peroxide in the product-inhibited state of MnSOD.     

Hydrogen peroxide triggers the proteolytic cleavage and the inactivation of calcineurin

Increases in the levels of reactive oxygen species (ROS) are correlated with a decrease in calcineurin (CN) activity under oxidative or neuropathological conditions. However, the molecular mechanism underlying this ROS-mediated CN inactivation remains unclear. Here, we describe a mechanism for the inactivation of CN by hydrogen peroxide. The treatment of mouse primary cortical neuron cells with Abeta(1-42) peptide and hydrogen peroxide triggered the proteolytic cleavage of CN and decreased its enzymatic activity. In addition, hydrogen peroxide was found to cleave CN in different types of cells. Calcium influx was not involved in CN inactivation during hydrogen peroxide-mediated cleavage, but CN cleavage was partially blocked by chloroquine, indicating that an unidentified lysosomal protease is probably involved in its hydrogen peroxide-mediated cleavage. Treatment with hydrogen peroxide triggered CN cleavage at a specific sequence within its catalytic domain, and the cleaved form of CN had no enzymatic ability to dephosphorylate nuclear factor in activated T cells. Thus, our findings suggest a molecular mechanism by which hydrogen peroxide inactivates CN by proteolysis in ROS-related diseases.     

Doxorubicin enhances complement susceptibility of human melanoma cells by extracellular oxygen radical formation

In two recent publications we showed that rapid inactivation of cell-bound C3b is a protective mechanism of human melanoma cells against killing by the R24 monoclonal antibody and human complement (Panneerselvam, M., Welt, S., Old, L.J., and Vogel, C.-W. (1986) J. Immunol. 136, 2534-2541) and that this protective mechanism can be inhibited by both the free and immobilized anthracycline glycoside doxorubicin (adriamycin) resulting in an enhanced complement susceptibility (Panneerselvam, M., Bredehorst, R., and Vogel, C.-W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9144-9148). In this paper we show that the complement enhancing effect of both free and immobilized doxorubicin is caused by the generation of reactive oxygen species including superoxide anion radical, hydrogen peroxide, and hydroxyl radical. The complement-enhancing effect of the anthracyclines can be completely inhibited by the reactive oxygen scavengers superoxide dismutase, catalase, and dimethyl sulfoxide. Consistent with this observation, 5-iminodaunorubicin, an anthracycline glycoside with an imine-substituted quinone moiety and, therefore, with a significantly reduced ability to form oxygen radicals, did not cause an enhanced-complement susceptibility. The complement-enhancing effect of the anthracycline glycosides could also be inhibited by bivalent metal chelators but was unaffected by sulfhydryl-blocking reagents or glutathione. Our results suggest that the anthracycline glycosides generate in a metal- (most probably iron) dependent reaction superoxide anion radicals with subsequent formation of hydrogen peroxide and hydroxyl radicals. These reactive oxygen species then cause alterations in the melanoma cells resulting in the enhanced complement susceptibility. While the target molecule(s) of the reactive oxygen species responsible for the enhanced complement susceptibility is not known, the data obtained with immobilized doxorubicin suggest that the target molecule(s) is located in the cell membrane.     

Modification of proteins by active oxygen and their degradation

A detailed analysis of literary data concerning the oxidative modification of proteins by active oxygen species was carried out. It was shown that intermediate products of molecular oxygen reduction, e.g., superoxide anion radical, hydrogen peroxide and hydroxyl radical, can induce the inactivation of enzymes in vitro as a result of oxidative modification of certain amino acid residues necessary for the maintenance of native properties of the enzyme. In some cases modification of enzymes results in their degradation by proteolytic enzymes. Besides, some enzymes catalyzing the interconversions of active oxygen species (catalase superoxide dismutase, cytochrome P-450) are also inactivated in the course of catalysis under the oxidative action of active oxygen species. It was assumed that the oxidative modification of proteins appears to be one of the mechanisms which control their degradation in the cell. The hydroxyl radical oxidizing the amino acid residues located in the vicinity of the site of its synthesis is a direct modifying species. The superoxide anion radical and hydrogen peroxide are hydroxyl radical precursors and are responsible for the transport of oxidizing equivalents in the cell.