Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.10.3.1 (tyrosinase)
9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The reaction of terminal oxidation of the substrate (catechol) by molecular oxygen catalyzed by o-diphenoloxidase (o-diphenol: oxygen oxydoreductase; EC 1.10.3.1) is found to occur via a free radical mechanism. The copper of the active center changes its valency during the reaction. The spectra of substrate radicals and of the Cu2+ ions were registered by means of a high sensitivity ESR-spectrometer and their concentrations were determined.
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PMID:[Mechanism of catalysis by o-diphenol oxidase]. 21 16

Evidence for the generation of superoxide anion in an enzymatic action of tyrosinase is reported. In the dopatyrosinase reaction, 1 mol of O2 is required for the production of 2 mol of dopaquinone, 1 mol of dopachrome, and 1/4 mol of O2-. Superoxide dismutase and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (a chemiluminescence probe and O2 trap) do not inhibit the rate of dopachrome formation from dopa in the presence of tyrosinase, indicating that free O2- is not utilized for metabolizing dopa. ESR studies for the accumulation of semiquinone radicals generated from tyrosine and N-acetyltyrosine in the presence of tyrosinase imply that O2- is not generated by the semiquinone + O2 reaction. Since the addition of H2O2 and dopa to tyrosinase promotes the release of O2- and formation of dopachrome, the Cu(II)O2-Cu(I) complex could be formed as a intermediate (an active form of tyrosinase); [Cu(II)]2 + H2O2 in equilibrium Cu(I)O2-Cu(II) + 2H+.
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PMID:Generation of superoxide during the enzymatic action of tyrosinase. 130 77

The alpha, beta, and gamma isozymes of Agaricus bisporus tyrosinase undergo inactivation in the presence of oxalate. The inactivation rate law is first order in enzyme and second order in oxalate. On a more rapid time scale than inactivation, oxalate acts as a competitive inhibitor of the catecholase reaction of tyrosinase. After removal of oxalate by dialysis, the inactivated enzyme is found to contain 50% of the original copper, all of which is present as paramagnetic, mononuclear copper sites. The ESR parameters of this copper indicate a tetragonal environment with nitrogen and oxygen ligands. The product of oxalate inactivation has lost one copper from each binuclear site and is thus a metapo derivative. Addition of Cu(II) to metapotyrosinase results in complete recovery of copper and catalytic activity. Prolonged storage of metapotyrosinase, in the absence of any additional Cu(II), results in copper migration, producing a 50% recovery of the original specific activity, expressed on a protein basis. Copper migration converts metapo sites into equal numbers of reconstituted, holo sites and fully apo sites. Both copper migration and copper reconstitution follow apparent first-order kinetics and are pH dependent. The involvement of two ionizable groups accounts for the observed pH dependence of each process. For copper migration pKa values of 6.0 and 8.8 were found, while for copper reconstitution the pKa values were 5.4 and 6.9. Addition of either Co(II) or Zn(II) to metapotyrosinase results in the formation of enzymatically inactive, mixed-metal derivatives of the binuclear copper site having one Cu(II) and one Co(II) or Zn(II) ion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Agaricus bisporus metapotyrosinase: preparation, characterization, and conversion to mixed-metal derivatives of the binuclear site. 217 54

The oxidation of four catechol(amine)s by tyrosinase has been studied by electron spin resonance and optical methods. Rates of oxygen consumption and of dopaquinone and dopachrome formation during the oxidation of dopa have been measured, and compared with rates of dopasemiquinone production measured using spin-stabilization procedures. In the presence of spin-stabilizing metal ions, production of semiquinone is approximately quantitative. Time-dependent ESR spectra obtained from dopa and dopamine show a slow regeneration of semiquinone, suggesting that a semiquinone precursor is slowly reformed. In contrast, time-dependent spectra for 4-methylcatechol and N-acetyldopamine show decay of the primary semiquinone together with buildup of a secondary semiquinone apparently derived from the corresponding 6-hydroxy-catechol(amine). Thus, catecholamines that give rise to a cyclizable quinone show a pattern of behavior that differs from those that produce a non-cyclizable quinone. These results are discussed in terms of their possible significance to melanogenesis and the toxicity of catechol(amine)s, which has been attributed to production of semiquinones and/or other oxygen radicals.
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PMID:Tyrosinase-catalyzed oxidation of dopa and related catechol(amine)s: a kinetic electron spin resonance investigation using spin-stabilization and spin label oximetry. 303 39

In the present work, we studied the effects of phenoxyl radicals, generated by tyrosinase-catalyzed oxidation of a phenolic antitumor drug, Etoposide (VP-16), on a purified dog kidney Na+/K(+)-ATPase by characterizing interactions of VP-16 phenoxyl radicals with the enzyme's SH-groups by ESR and correlating the loss of the enzymatic activity with the oxidation of its SH-groups, and oxidation of VP-16. VP-16/tyrosinase caused inhibition of Na+/K(+)-ATPase which was dependent on the incubation time and concentration of tyrosinase. The inhibition of Na+/K(+)-ATPase was accompanied by a decrease of DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)-titratable SH-groups. In the presence of Na+/K(+)-ATPase, a typical ESR signal of the VP-16 phenoxyl radical could be observed only following a lag period the duration of which was proportional to the concentration of the Na+/K(+)-ATPase added. Our HPLC measurements demonstrated that Na+/K(+)-ATPase protected VP-16 against tyrosinase-catalyzed oxidation. Combined these results suggest that redox-cycling of VP-16/VP-16 phenoxyl radical by SH-groups of Na+/K(+)-ATPase occurred. Ascorbate which is known to reduce the VP-16 phenoxyl radicals, protected the enzyme against inactivation, prevented oxidation of the enzyme's SH-groups. Reduction of VP-16 phenoxyl radicals by ascorbate was directly observed by the semidehydroascorbyl radical signal in the ESR spectra. VP-16 phenoxyl radical-induced oxidation of sulfhydryls and inhibition of the Na+/K(+)-ATPase may be responsible for at least some of its clinical side effects (e.g., cardiotoxicity) which can be prevented by ascorbate.
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PMID:Inhibition of Na+/K(+)-ATPase by phenoxyl radicals of etoposide (VP-16): role of sulfhydryls oxidation. 749 37

Phenolic compounds can act as radical scavengers due to their ability to donate a mobile hydrogen to peroxyl radicals producing a phenoxyl radical if the phenoxyl radical formed in the radical scavenging reaction efficiently interacts with vitally important biomolecules, then this interaction may result in cytotoxic effects rather than in antioxidant protection. In the present work we have chosen two model compounds--a phenolic antitumor drug, VP-16, known to be highly cytotoxic, and a homolog of vitamin E, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC)--as typical representatives of phenoxyl radicals to study interactions of their phenoxyl radicals with intracellular thiols. Using a water-soluble source of peroxyl radicals, the azo-initiator 2,2'-azobis(2-aminodinopropane) (AAPH), we found that both PMC and VP-16 are very efficient scavengers of peroxyl radicals as evidenced by their ability to inhibit AAPH-induced chemiluminescence of luminol and oxidation of PnA incorporated into DOPC liposomes. Both PMC and VP-16 were also able to protect against AAPH-induced oxidative degradation of DNA in nuclei from human leukemic K562 cells. In contrast, there was a dramatic difference in the ability of VP-16 and PMC to protect GSH against AAPH-induced oxidation: while PMC inhibited AAPH-induced oxidation of GSH in a concentration-dependent manner, VP-16 did not protect GSH against oxidation. We hypothesized that this was due to different reactivities of the phenoxyl radicals formed by AAPH-derived peroxyl radicals from VP-16 and PMC toward GSH. To substantiate this hypothesis, we compared interactions of the phenoxyl radicals generated from VP-16 and PMC with intracellular thiols in K562 cell homogenates. While the PMC phenoxyl radicals were only slightly affected by thiols, the VP-16 phenoxyl radicals were reduced by thiols. This is evidenced by (i) a significant inhibition of the tyrosinase-induced VP-16 consumption upon addition of K562 cell homogenates, (ii) a depletion of endogenous thiols in K562 cell homogenates induced by VP-16+tyrosinase, (iii) a transient disappearance of the VP-16 phenoxyl radical signal from the ESR spectra and its reappearance after depletion of endogenous thiols, and (iv) elimination of the lag period for the appearance of the VP-16 phenoxyl radical ESR signal subsequent to depletion of thiols by mersalyl acid. To evaluate the contribution of GSH and protein thiols to reduction of the VP-GSH-peroxidase + cumeme hydroperoxide to specifically deplete endogenous GSH.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Phenoxyl radicals of etoposide (VP-16) can directly oxidize intracellular thiols: protective versus damaging effects of phenolic antioxidants. 771 69

Mechanisms of phenoxyl radical-induced generation of oxygen radicals potentially involved in toxicity of benzene were studied. We hypothesized that phenoxyl radical intermediates formed from phenolic metabolites of benzene by oxidative enzymes (e.g., peroxidases, tyrosinase) are able to damage biomolecules via (i) oxidation of low-molecular-weight thiols and protein thiols and (ii) thiol-dependent generation of oxygen radicals and subsequent oxidation of DNA. Phenoxyl radicals were generated by the oxidation of phenol by myeloperoxidase+H2O2, horseradish peroxidase+H2O2, or tyrosinase. The reaction of phenolphenoxyl radicals with GSH and dihydrolipoic acid was studied. Our HPLC measurements showed that both thiols reduced the phenoxyl radical back to phenol. This reaction was accompanied by the formation of thiyl radicals (detected by ESR as 5,5-dimethyl-1-pyrroline-N-oxide/glutathione thiyl radical spin adducts) and of superoxide radicals (measured by their chemiluminescence response in the presence of lucigenin). Hydroxylation of 2'-deoxyguanosine to 8-oxo-7,8-dihydro-2'-deoxyguanosine was demonstrated in the course of the tyrosinase-catalyzed oxidation of phenol in the presence of dihydrolipoic acid and Fe(III)-EDTA. Redox-cycling of phenoxyl radicals by thiols produces oxygen radicals which can be responsible for the oxidative damage of DNA by radical intermediates of benzene metabolism.
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PMID:Phenoxyl radical-induced thiol-dependent generation of reactive oxygen species: implications for benzene toxicity. 789 44

Etoposide (VP-16) is an antitumor drug currently in use for the treatment of a number of human cancers. Mechanisms of VP-16 cytotoxicity involve DNA breakage secondary to inhibition of DNA topoisomerase II and/or direct drug-induced DNA strand cleavage. The VP-16 molecule contains a hindered phenolic group which is crucial for its antitumor activity because its oxidation yields reactive metabolites (quinones) capable of irreversible binding to macromolecular targets. VP-16 phenoxyl radical is an essential intermediate in VP-16 oxidative activation and can be either converted to oxidation products or reduced by intracellular reductants to its initial phenolic form. In the present paper we demonstrate that the tyrosinase-induced VP-16 phenoxyl radical could be reduced by ascorbate, glutathione (GSH) and dihydrolipoic acid. These reductants caused a transient disappearance of a characteristic VP-16 phenoxyl radical ESR signal which reappeared after depletion of the reductant. The reductants completely prevented VP-16 oxidation by tyrosinase during the lag-period as measured by high performance liquid chromatography; after the lag-period VP-16 oxidation proceeded with the rate observed in the absence of reductants. In homogenates of human K562 leukemic cells, the tyrosinase-induced VP-16 phenoxyl radical ESR signal could be observed only after a lag-period whose duration was dependent on cell concentration; VP-16 oxidation proceeded in cell homogenates after this lag-period. In homogenates of isolated nuclei, the VP-16 phenoxyl radical and VP-16 oxidation were also detected after a lag-period, which was significantly shorter than that observed for an equivalent amount of cells. In both cell homogenates and in nuclear homogenates, the duration of the lag period could be increased by exogenously added reductants. The duration of the lag-period for the appearance of the VP-16 phenoxyl radical signal in the ESR spectrum can be used as a convenient measure of cellular reductive capacity. Interaction of the VP-16 phenoxyl radical with intracellular reductants may be critical for its metabolic activation and cytotoxic effects.
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PMID:Tyrosinase-induced phenoxyl radicals of etoposide (VP-16): interaction with reductants in model systems, K562 leukemic cell and nuclear homogenates. 816 27

Sodium 5,6-benzylidene ascorbate (SBA) is a conjugate of ascorbic acid (Asc) with benzyaldehyde. It has been found that the antioxidant activity of SBA is more stable and has a longer lifetime in living cells and organs than Asc. In this study, we investigated the effect of SBA on the induction of melanin in cultured melanoma (B-16) cells irradiated by UV-A. Melanin content of B-16 cells was significantly increased by UV-A irradiation. The induction was abolished by mannitol and particularly by superoxide dismutase, suggesting the involvement of O2- in the biosynthesis of melanin in cultured melanoma cells. This was theorized by the fact that the induction was also observed in B-16 cells treated with superoxide anion radicals chemically generated in the hypoxanthine/xanthine oxidase-reaction system, instead of UV-A irradiation. The induction of melanin caused by UV-A irradiation was suppressed by SBA in a dose-dependent manner. To elucidate the mechanism of this suppressive effect, the scavenging activity against O2-, and the inhibitory effect of SBA on tyrosinase activity were examined. ESR spectrometric analysis showed that SBA strongly scavenged O2-, and the presence of SBA in the medium remarkably inhibited the tyrosinase activity in cultured B-16 melanoma cells. It can be concluded that SBA effectively inhibits the melanin biosynthesis in B-16 melanoma cells induced by reactive oxygen species (ROS) generated by UV-A irradiation via tyrosinase.
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PMID:Inhibitory effect of sodium 5,6-benzylidene ascorbate (SBA) on the elevation of melanin biosynthesis induced by ultraviolet-A (UV-A) light in cultured B-16 melanoma cells. 853 99

Some studies have shown the potential relevance of the oxidation products of 4-hydroxytamoxifen (4OHTAM) in carcinogenesis. Other studies show 4OHTAM has antioxidant properties. We characterized the one-electron oxidative activation reactions of 4OHTAM and three other phenolics, 3-hydroxytamoxifen (3OHTAM), 1-(4-hydroxyphenyl)-1, 2-diphenylethene, and phenol (PhOH), catalyzed by myeloperoxidase (MPx), horseradish peroxidase (HRP), lactoperoxidase, mushroom tyrosinase, and nonenzymatic initiators in vitro under a variety of conditions and in cells. Differences in activation of the phenolics by the enzymes were directly compared using cis-parinaric acid (PnA)-loaded human serum albumin. All phenolics were substrates for the enzymes, but MPx only weakly activated 4OHTAM to its phenoxyl radical. In HL60 cells loaded metabolically with PnA so that effects on phospholipids could be monitored by HPLC with fluorescence detection, PhOH plus H2O2 caused massive oxidation across all phospholipid classes. 4OHTAM dose-dependently protected phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine against both H2O2-induced and normal metabolic oxidation. This suggested 4OHTAM is a poor substrate for intracellular MPx. In rat aorta smooth muscle cells loaded with PnA, 4OHTAM also protected against AMVN-induced peroxidation of those three phospholipids and sphingomyelin, whereas 3OHTAM did not. Spin trapping of glutathionyl radicals (GS*) with DMPO and quantifying the ESR-silent nitrone form of the GS-DMPO adduct by HPLC showed that neither 3OHTAM plus H2O2 nor 4OHTAM plus H2O2 caused a significant level of GSH oxidation with isolated MPx, nor did the latter in HL60 cells, whereas PhOH plus H2O2 was a potent source of GS* in both systems. Both 4OHTAM and 3OHTAM formed the nitrone adduct under cell-free conditions when activated with HRP. The data show that the substrate specificity of a given (myelo)peroxidase determines if a phenolic exerts pro- (through generation of reactive phenoxyl radicals) or antioxidant (through radical scavenging) properties in intracellular environments.
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PMID:Peroxidase-catalyzed pro- versus antioxidant effects of 4-hydroxytamoxifen: enzyme specificity and biochemical sequelae. 989 15


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