Sarcoma de Kaposi de tipo linfangiomatoso / Lymphangioma-like Kaposi sarcoma

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Tyrosinase action on monophenols: evidence for direct enzymatic release of o-diphenol.

Using gas chromatography-mass spectrometry, the direct enzymatic release of o-diphenol (4-tert-butylcatechol) during the action of tyrosinase on a monophenol (4-tert-butylphenol) has been demonstrated for the first time in the literature. The findings confirm the previously proposed mechanism to explain the action of tyrosinase on monophenols (J.N. Rodríguez-López, J. Tudela, R. Varón, F. García-Carmona, F. García-Cánovas, J. Biol. Chem. 267 (1992)). Oxytyrosinase, the oxidized form of the enzyme with a peroxide group, is the only form capable of catalysing the transformation of monophenols into diphenols, giving rise to an enzyme-substrate complex in the process. The o-diphenol formed is then released from the enzyme-substrate complex or oxidized to the corresponding o-quinone. In order to detect the enzymatic release of o-diphenol, the non-enzymatic evolution of the o-quinone to generate o-diphenol by weak nucleophilic attack reactions and subsequent oxidation-reduction was blocked by the nucleophilic attack of an excess of cysteine. Furthermore, the addition of catalytic quantities of an auxiliary o-diphenol (e.g. catechol) considerably increases the accumulation of 4-tert-butylcatechol. The enzyme acting on 4-tert-butylphenol generates the enzyme-4-tert-butylcatechol complex and 4-tert-butylcatechol is then released (with k(-2)) generating mettyrosinase. The auxiliary o-diphenol added (catechol) and the 4-tert-butylcatechol generated by the enzyme then enter into competition. When [catechol] >> [4-tert-butylcatechol], the enzyme preferentially binds with the catechol to close the catalytic cycle, while 4-tert-butylcatechol is accumulated in the medium. In conclusion, we demonstrate that the enzyme produces 4-tert-butylcatechol from 4-tert-butylphenol, the concentration of which increases considerably in the presence of an auxiliary o-diphenol such as catechol.

Catalytic oxidation of 2,4,5-trihydroxyphenylalanine by tyrosinase: identification and evolution of intermediates.

The oxidation of 3,4-dihydroxyphenylalanine (dopa) by O2 catalyzed by tyrosinase yields 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaquinone-H+). This evolves non-enzymatically through two branches (cyclization and/or hydroxylation), whose respective operations are determined by pH. The hydroxylation branch of o-dopaquinone-H+ only operates significantly at pH < or = 5.0 and involves the accumulation of 2,4,5-trihydroxyphenylalanine (topa), which has been detected by high-performance liquid chromatography (HPLC). This last compound is also a substrate of tyrosinase. The oxidation of topa by both tyrosinase and periodate yields 5-(2-carboxy-2-aminoethyl)-4-hydroxy-1,2-benzoquinone, with its amino group protonated (o-topaquinone-H+), which is red (RTQH) (lambda max 272-485 nm) at pH 7.0 and yellow (TTQH) (lambda max 265-390 nm) at pH 3.0. This is based on pKa 4.5 of the 2-OH group of the benzene ring of o-topaquinone-H+, as derived from spectrophotometric and HPLC assays. At physiological pH, RTQH undergoes deprotonation of the ammonium group of the side chain to yields RTQ, which cyclize into 2-carboxy-2,3-dihydroxyindolen-5,6-quinone (dopachrome), with a 1:1 stoichiometry and first-order kinetics. The evolution of RTQH has been analyzed by spectrophotometry, HPLC, cyclic voltammetry and constant potential electrolytic assays. From HPLC assays, the value of the first-order constant for the evolution of RTQH at pH 7.0 (kRTQHapp 4.83 x 10(-5) s-1), as well as of the rate constant for the cyclization step of RTQ (kRTQc 2.53 x 10(-3) s-1) were determined.

Effect of pH on the oxidation pathway of dopamine catalyzed by tyrosinase.

The oxidation of 3,4-dihydroxyphenylethylamine (dopamine) by O2 catalyzed by tyrosinase yields 4-(2-aminoethyl)-1, 2-benzoquinone (o-dopaminequinone), which evolves nonenzymatically through two branches or sequences of reactions, whose respective operations are determined by the pH of the medium. The cyclization branch of o-dopaminequinone takes place in the entire range of pH and is the only significant branch at pH greater than or equal to 6. The hydroxylation branch of o-dopaminequinone only operates significantly at pH less than 6, and involves the accumulation of 2,4,5-trihydroxyphenylethylamine (6-hydroxydopamine) and 5-(2-aminoethyl)-2-hydroxy-1,4-benzoquinone (p-topaminequinone), identified from cyclic voltammetry assays. The kinetic characterization of the hydroxylation branch of o-dopaminequinone has been carried out by spectrophotometric and oxymetric assays. The successful fitting of data to the kinetic behavior predicted by the kinetic analysis at both pH greater than or equal to 6 and pH less than 6 confirms the overall oxidation pathway proposed for the dopamine oxidation catalyzed by tyrosinase. The antitumoral power of dopamine is possibly enhanced by the high cytotoxicity of 6-hydroxydopamine and p-topaminequinone, accumulated at the acidic pH characteristic of melanosomes and melanome cells.

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.