A sensitive new assay for the oxidation of 3,4-dihydroxy-l-phenylalanine by tyrosinase

A sensitive new assay for the oxidation of 3,4-dihydroxy-l-phenylalanine (dopa) employs [2,5,6-³H]dopa and involves the measurement of ³H⁺ released from the 6-position when the dihydroindole ring is formed. At its lower limit of sensitivity the assay will measure the oxidation of about 0.8 nmol min⁻¹ of dopa and also permits the measurement of this activity in homogenates and turbid solutions. Use of the radiometric assay proves that the slow step in the overall conversion of dopa to dopachrome is the oxidation of 2,3-dihydro-5,6-dihydroxyindole-2-carboxylate to dopachrome.     

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.     

Enkephalins and exorphins oxidation by tyrosinase

Tyrosinase activity was tested on some tyrosine-containing peptides (enkephalins and exorphins). All they are substrates for tyrosinase, showing a good affinity for the enzyme, in some cases higher than tyrosine itself. Aminoacid analysis after hydrolysis of long-lasting incubation mixtures of tyrosinase with Leu-enkephalin in presence of reductants demonstrates the formation of DOPA. The production of a new peptide containing DOPA derived from the oxidation of Leu-enkephalin was revealed by high performance liquid chromatography (HPLC).     

Chemical intermediates in dopamine oxidation by tyrosinase,and kinetic studies of the process

A minor pathway for dopamine oxidation to dopaminochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometric determination have both been undertaken. After oxidizing dopamine with mushroom tyrosinase or sodium periodate in a pH range from 6.0 to 7.0, it was spectrophotometrically possible to detect o-dopaminoquinone-H⁺ as the first intermediate in this pathway. The steps for dopamine transformation to dopaminochrome are as follows: dopamine → o-dopaminequinone-H⁺ → o-dopaminequinone → leuko-dopaminochrome → dopaminochrome. No participation of oxygen was detected in the conversion of leukodopaminochrome to dopaminochrome. Scanning spectroscopy and graphical analysis of the obtained spectra also verified that dopaminequinone-H⁺ was transformed into aminochrome in a constant ratio. The stoichiometry equation for this conversion is 2 o-dopaminequinone-H⁺ → dopamine + dopaminochrome. The pathway for dopamine oxidation to dopaminochrome by tyrosinase has been studied as a system of various chemical reactions coupled to an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system; this type of mechanism has been named “Enzymatic-chemical-chemical” (E_ZCC). Rate constants for the implied chemical steps at different pH and temperature values have been evaluated from the measurement of the lag period arising from the accumulation of dopaminochrome that took place when dopamine was oxidized at acid pH. The thermodynamic activation parameters of the chemical steps, the deprotonation of dopaminequinone-H⁺ to dopaminequinone, and the internal cyclization of dopaminequinone to leukodopaminochrome have been calculated.     

Study of alpha-methyldopa oxidation by tyrosinase

The pathway for alpha-methyldopa oxidation to alpha-methyldopachrome, by mushroom tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been undertaken. The steps for alpha-methyldopa transformation into its aminochrome would be: alpha-methyldopa----o-alpha-methyldopaquinone-H+----o-alpha- methyldopaquinone----leuko-alpha-methyldopachrome----alpha- methyldopachrome. The stoichiometry for this conversion corresponded to the equation: 2 o-alpha-methyldopaquinone-H+----alpha-methyldopa + alpha-methyldopachrome. At very acid pH values, another route implying the addition of water to the quinonic ring, competes with the first one. Two chemical pathways can be proposed from alpha-methyldopaquinone-H+, the relative importance of which is determined by the pH. A theoretical and experimental kinetic approach was applied to this oxidative reaction. Rate constants and thermodynamic activation parameters of the chemical steps, have been evaluated. The results obtained confirmed that alpha-methyldopa oxidation by tyrosinase followed a scheme similar to that established for L-dopa and alpha-methylnoradrenaline.     

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.     

Uridine diphosphate 2-deoxyglucose: Chemical synthesis,enzymic oxidation and epimerization

The paper describes chemical synthesis of uridine diphosphate 2-deoxyglucose (UDPdGlc) through reaction of uridine 5′-phosphomorpholidate with 2-deoxy-α-d-glucopyranosyl phosphate. The prepared analog of uridine diphosphate glucose (UDPGlc) served as a substrate for calf liver UDPGlc dehydrogenase (EC 1.1.1.22), the reaction product was identified as nucleotide deoxyhexuronic acid derivative. The apparent K_m for UDPdGlc was found to be 60 times that of UDPGlc, and the relative V value for the analog was 0.09. The peculiar lag-period in reaction kinetics has been observed for the analog, and is presumably connected with the slow rate of the initial stages of the reaction. UDPdGlc was found to be quite an efficient substrate for UDPGlc 4-epimerases (EC 5.1.3.2) from yeast, calf liver and mung bean seedlings.     

Oxidation of tyrosine residues in proteins by tyrosinase. Formation of protein-bonded 3,4-dihydroxyphenylalanine and 5-S-cysteinyl-3,4-dihydroxyphenylalanine.

A simple and rapid method was developed for the determination of 3,4-dihydroxyphenylalanine (dopa) and 5-S-cysteinyl-3,4-dihydroxyphenylalanine (5-S-cysteinyldopa) in proteins with the use of high-pressure liquid chromatography. With this method, it is demonstrated that mushroom tyrosinase can catalyse hydroxylation of tyrosine residues in proteins to dopa and subsequent oxidation to dopaquinone residues. The dopaquinone residues in proteins combine with cysteine residues to form 5-S-cysteinyldopa in bovine serum albumin and yeast alcohol dehydrogenase, whereas dopa is the major product in bovine insulin, which lacks cysteine residues.     

Indirect oxidation of 6-tetrahydrobiopterin by tyrosinase

6-Tetrahydrobiopterin is known to bind to an allosteric site of tyrosinase to directly inhibit the enzyme. However, simultaneous measurements of ultraviolet-visible absorption spectra and oxygen consumption led us to conclude that the inhibition was due to oxidation of 6-tetrahydrobiopterin by dopaquinone. Immediately after addition of 6-tetrahydrobiopterin, tyrosinase stopped producing dopachrome from either tyrosine or dopa. Duration of inhibition was proportional to the concentration of added 6-tetrahydrobiopterin and the enzyme activity was fully restored after the inhibition. Surprisingly, there was a rapid consumption of oxygen during the inhibition period. In addition, absorption spectra indicated that the only reaction that occurred during the inhibition was oxidation of 6-tetrahydrobiopterin to 7,8-dihydrobiopterin. In the absence of tyrosine or dopa, tyrosinase did not oxidize 6-tetrahydrobiopterin, suggesting that a reaction intermediate between dopa and dopachrome was a target for the inhibition. We propose a new mechanism in which dopa is oxidized to dopaquinone and the latter, instead of producing dopachrome, is reduced back to dopa by 6-tetrahydrobiopterin.     

Photochemical oxidation of tyrosinase

Dye sensitized photo-oxidation inactivates tyrosinases isolated from Neurospora and Agaricus. The rate of inactivation is enhanced by cyanide and is dependent on pH.     

Kinetic study of sinephrine oxidation by mushroom tyrosinase

Mushroom tyrosinase catalyzes the oxidation of sinephrine showing a marked lag period during appearance of adrenochrome and simultaneously adrenaline accumulation in the reaction medium can be detected. The adrenaline accumulation follows a sigmoidal curve until a constant level of adrenaline is reached when the system is in the steady-state. These experimental results agree with a model of enzymatic catalysis that includes the chemical evolution of adrenoquinone and permit us to explain these phenomenon as well as the influence that enzyme and sinephrine concentration present on the lag period and the level of adrenaline accumulated in the steady-state.     

Catalytic oxidation of o-aminophenols and aromatic amines by mushroom tyrosinase

The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied. The catalytic constants of aromatic monoamines and o-diamines were both low, these results are consistent with our previous mechanism in which the slow step is the transfer of a proton by a hydroxyl to the peroxide in oxy-tyrosinase (Fenoll et al., Biochem. J. 380 (2004) 643-650). In the case of o-aminophenols, the hydroxyl group indirectly cooperates in the transfer of the proton and consequently the catalytic constants in the action of tyrosinase on these compounds are higher. In the case of aromatic monoamines, the Michaelis constants are of the same order of magnitude than for monophenols, which suggests that the monophenols bind better (higher binding constant) to the enzyme to facilitate the π-π interactions between the aromatic ring and a possible histidine of the active site. In the case of aromatic o-diamines, both the catalytic and Michaelis constants are low, the values of the catalytic constants being lower than those of the corresponding o-diphenols. The values of the Michaelis constants of the aromatic o-diamines are slightly lower than those of their corresponding o-diphenols, confirming that the aromatic o-diamines bind less well (lower binding constant) to the enzyme.     

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.     

The formation of dihydrodiols by chemical or enzymic oxidation of 3-methylcholanthrene.

The chemical oxidation of 3-methylcholanthrene in an ascorbic acid-ferrous sulphate-EDTA reaction mixture gave all five possible dihydrodiols. The structures and stereochemistry of the dihydrodiols were shown by UV, mass and NMR spectral studies and by chemical examination to be cis-2a,3-dihydroxy-3-methylcholanthrene, trans-4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene, cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. An examination by HPLC of the dihydrodiols formed in the metabolism of 3-methylcholanthrene by rat-liver microsomal preparations showed the presence of trans-4,5-dihydro-4,5-dihydoxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, identified by comparison of their UV and chromatographic characteristics with those of authentic standards. Tentative identification of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, cis-2a,3-dihydroxy-3-methylcholanthrene and cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene as metabolites were made from their mobilities using HPLC. A quantitative comparison of the dihydrodiols formed from 3H-labelled 3-methylcholanthrene by microsomal preparations from the livers of normal and 3-methylcholanthrene-treated rats was carried out. trans-9,10-Dihydro-9,10-dihydroxy-3-methylcholanthrene and cis- and trans-1,2-dihydroxy-3-methylcholanthrene were formed when 3-methylcholanthrene was incubated with mouse skin in organ culture.