Oxidation of 6-hydroxydopamine catalyzed by tyrosinase

• 1.1. The oxidation of 3,4-dihydroxyphenylethylamine (dopamine) by O₂ catalyzed by tyrosinase yields 4-(2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaminequinone-H⁺, which evolves non-enzymatically through two branches or sequences of reactions, whose respective operations are determined by the pH of the medium.
• 2.2. The cyclization branch of o-dopaminequinone-H⁺ takes place in the entire range of pH and is the only significant branch at pH ⩾ 6.
• 3.3. The hydroxylation branch of o-dopaminequinone-H⁺ only operates significantly at pH < 6, and involves the accumulation of 2,4,5-trihydroxyphenylethylamine (6-hydroxydopamine), identified by high performance liquid chromatography (HPLC).
• 4.4. 6-hydroxydopamine is also a substrate of tyrosinase. The identification and evolution of the oxidation products of 6-hydroxydopamine has been carried out by spectrophotometry and HPLC assays.

     

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 role of pH in the melanin biosynthesis pathway

Having oxidized 3,4-dihydroxyphenylalanine (dopa) with sodium periodate or mushroom tyrosinase in a pH range from 3.5 to 6.0, it has been possible to detect spectrophotometrically 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone with the amino group protonated (o-dopaquinone-H+), a postulated intermediate in the melanogenesis pathway. When the pH value was greater than 4, the final product obtained was 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome); however, for pH values lower than 4, two different products were identified by means of cyclic voltammetry: 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone and dopachrome. These products appeared when oxidation was achieved with the enzyme as well as with periodate. This suggests that two chemical pathways can arise from alpha-dopaquinone-H+, whose relative importance is determined by the pH. The steps of these pathways would be dopa leads to o-dopaquinone-H+ leads to o-dopaquinone leads to leukodopachrome leads to dopachrome, for the first one, and dopa leads to o-dopaquinone-H+ leads to 2,4,5-trihydroxyphenylalanine leads to 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone very slowly leads to intermediate compound leads to dopachrome, for the second one. The stoichiometry for the conversion of dopaquinone-H+ into dopachrome for pH values greater than 4 followed equation of 2 o-dopaquinone-H+ leads to dopa + dopachrome. No participation of oxygen was detected in the conversion of leukodopachrome (2,3-dihydro-5,6-dihydroxyindole-2-carboxylate) into dopachrome.     

The role of 2,4,5-trihydroxyphenylalanine in melanin biosynthesis.

Both 3,4-dihydroxyphenylalanine and 2,4,5-trihydroxyphenylalanine were oxidized with periodate and mushroom tyrosinase to determine whether the latter compound is an intermediate in melanin biosynthesis. Matrix analysis of the spectra obtained with a rapid scan spectrophotometer and comparison of the spectra of quinone intermediates with model quinones disclosed that, although 2,4,5-trihydroxyphenylalanine can be oxidized to 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome), this oxidation proceeds through a stable intermediate, 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone, which does not appear in the oxidation of 3,4-dihydroxyphenylalanine to dopachrome. Thus, these studies are in agreement with the original postulate, that 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone and leukodopachrome are the intermediates in the major pathway for dopachrome synthesis.     

Synthesis of gamma-L-glutaminyl-[3,5-3H]4-hydroxybenzene and the study of reactions catalyzed by the tyrosinase of Agaricus bisporus.

gamma-L-Glutaminyl-[3,5-3H]4-hydroxybenzene was synthesized in order to study the kinetics of its hydroxylation by tyrosinase purified from Agaricus bisporus and to explore its role in the induction of the dormant state in the spores of this species. It was found to be unique among the monophenolic substrates for tyrosinase in that the lag period for the hydroxylation reaction decreased with increasing substrate concentration. Unlike previously studied compounds, this phenol appeared to function as an electron donor, allowing it to act as its own co-substrate in the hydroxylation reaction. Its catechol product, gamma-L-glutaminyl-3,4-dihydroxybenzene, was found to be a superior co-substrate, yielding its electrons more readily (oxidation peak potential +0.18 V as compared with +0.65 V for the phenol). In situ periodate oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone confirmed the co-substrate role of the catechol in the hydroxylation reaction. The tyrosinase-mediated oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone occurred with an apparent Km = 1.54 mM and Vmax = 0.36 mmol/min/mg of enzyme. gamma-L-Glutaminyl-4-hydroxybenzene acted as an inhibitor of the oxidation reaction.     

Transplantation of Melanocytes Obtained from the Skin Ameliorates Apomorphine-Induced Abnormal Behavior in Rodent Hemi-Parkinsonian Models

Tyrosinase, which catalyzes both the hydroxylation of tyrosine and consequent oxidation of L-DOPA to form melanin in melanocytes, is also expressed in the brain, and oxidizes L-DOPA and dopamine. Replacement of dopamine synthesis by tyrosinase was reported in tyrosine hydroxylase null mice. To examine the potential benefits of autograft cell transplantation for patients with Parkinson’s disease, tyrosinase-producing cells including melanocytes, were transplanted into the striatum of hemi-parkinsonian model rats or mice lesioned with 6-hydroxydopamine. Marked improvement in apomorphine-induced rotation was noted at day 40 after intrastriatal melanoma cell transplantation. Transplantation of tyrosinase cDNA-transfected hepatoma cells, which constitutively produce L-DOPA, resulted in marked amelioration of the asymmetric apomorphine-induced rotation in hemi-parkinsonian mice and the effect was present up to 2 months. Moreover, parkinsonian mice transplanted with melanocytes from the back skin of black newborn mice, but not from albino mice, showed marked improvement in the apomorphine-induced rotation behavior up to 3 months after the transplantation. Dopamine-positive signals were seen around the surviving transplants in these experiments. Taken together with previous studies showing dopamine synthesis and metabolism by tyrosinase, these results highlight therapeutic potential of intrastriatal autograft cell transplantation of melanocytes in patients with Parkinson’s disease.     

Inhibitory effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase

Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay was performed in air-saturated solutions and the kinetic behavior of this enzyme in the oxidation of L-tyrosine and L-DOPA has been studied. The effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase have been studied. The results show that cupferron can inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag phase of tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activity of the enzyme decreased sharply. Cupferron can lead to reversible inhibition of the enzyme, possibly by chelating copper at the active site of the enzyme. The IC(50) value was estimated as 0.52 microM for monophenolase and 0.84 microM for diphenolase. A kinetic analysis shows that the cupferron is a competitive inhibitor for both monophenolase and diphenolase. The apparent inhibition constant for cupferron binding with free enzyme has been determined to be 0.20 microM for monophenolase and 0.48 microM for diphenolase.     

Isotopically labeled chlorobenzenes as probes for the mechanism of cytochrome P-450 catalyzed aromatic hydroxylation

Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates.     

Hydroxylating activity of tyrosinase and its dependence on hydrogen peroxide

The aim of this work was to study the hydroxylation of N, N-dimethyltyramine (DMTA) by tyrosinase in the presence of hydrogen peroxide, a reaction that does not take place without the addition of the hydrogen peroxide. Some properties of this hydroxylating activity are analyzed. The kinetic parameters of mushroom tyrosinase toward hydrogen peroxide (K(m) = 0.5 mM, V(m) = 11 microM/min, V(m)/K(m) = 2.2 x 10(-2) min(-1)) and toward DMTA (K(m) = 0.3 mM, V(m) = 4.8 microM/min, V(m)/K(m) = 16 x 10(-2) min(-1)) were evaluated. There was a lag period, which was similar to the characteristic lag of monophenolase activity at the expense of molecular oxygen. The length of this lag phase decreased with increasing hydrogen peroxide concentration, and disappeared at approximately 0.5 mM H(2)O(2). However, the lag was longer with higher DMTA concentrations. The pH optimum range for this hydroxylating activity was 6.0 to 7.0. The lag also varied with pH, increasing at pH values higher than 6.7. The presence of hydrogen peroxide is necessary for the oxidation of DMTA, as is the presence of active enzyme since the reaction was completely inhibited when selective tyrosinase inhibitors were added.     

Oxidation of 2,4-dichlorophenol catalyzed by horseradish peroxidase: characterization of the reaction mechanism by UV-visible spectroscopy and mass spectrometry

The hydrogen peroxide-oxidation of 2,4-dichlorophenol catalyzed by horseradish peroxidase has been studied by means of UV-visible spectroscopy and mass spectrometry in order to clarify the reaction mechanism. The dimerization of 2,4-dichlorophenol to 2,4-dichloro-6-(2,4-dichlorophenoxy)-phenol and its subsequent oxidation to 2-chloro-6-(2,4-dichlorophenoxy)-1,4-benzoquinone together with chloride release were observed. The reaction rate was found to be pH-dependent and to be influenced by the pK(a) value of 2,4-dichlorophenol. The dissociation constants of the 2,4-dichlorophenol/horseradish peroxidase (HRP) adduct at pH 5.5 and 8.5 were also determined: their values indicate the unusual stability of the adduct at pH 5.5 with respect to several adducts of HRP with substituted phenols.     

Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

A continuous spectrophotometric assay for determination of the aureusidin synthase activity of tyrosinase

Introduction – Aurones (aureusidin glycosides) are plant flavonoids that provide yellow colour to the flowers of some ornamental plants. In this study we analyse the capacity of tyrosinase to catalyse the synthesis of aureusidin by tyrosinase from the chalcone THC (2′,4′,6′,4–tetrahydroxychalcone). Objective – To develop a simple continuous spectrophotometric assay for the analysis of the spectrophotometric and kinetic characteristics of THC oxidation by tyrosinase. Methodology – THC oxidation was routinely assayed by measuring the increase in absorbance at 415 nm vs. reaction time. Results – According to the mechanism proposed for tyrosinase, the enzymatic reaction involves the o‐hydroxylation of the monophenol THC to the o‐diphenol (PHC, 2′,4′,6′,3,4 – pentahydroxychalcone), which is then oxidised to the corresponding o‐quinone in a second enzymatic step. This product is highly unstable and thus undergoes a series of fast chemical reactions to produce aureusidin. In these experimental conditions, the optimum pH for THC oxidation is 4.5. The progress curves obtained for THC oxidation showed the appearance of a lag period. The following kinetic parameters were also determined: K_m = 0.12 mM, V_m = 13 μM/min, V_m/K_m = 0.11/min. Conclusion – This method has made it possible to analyse the spectrophotometric and kinetic characteristics of THC by tyrosinase. This procedure has the advantages of a short analysis time, straightforward measurement techniques and reproducibility. In addition, it also allows the study of tyrosinase inhibitors, such as tropolone. Copyright © 2009 John Wiley & Sons, Ltd.     

Tyrosinase inhibition kinetics of anisic acid

Anisic acid (p-methoxybenzoic acid) was characterized as a tyrosinase inhibitor from ani-seed, a common food spice. It inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase with an IC50 of 0.60 mM. The inhibition of tyrosinase by anisic acid is a reversible reaction with residual enzyme activity. This phenolic acid was found to be a classical noncompetitive inhibitor and the inhibition constant K(I) was obtained as 0.603 mM. Anisic acid also inhibited the hydroxylation of L-tyrosine catalyzed by tyrosinase. The lag phase caused by the monophenolase activity was lengthened and the steady-state activity of the enzyme was decreased by anisic acid.     

Calculating molar absorptivities for quinones: application to the measurement of tyrosinase activity

The molar absorptivities of the quinones produced from different o-diphenols, triphenols, and flavonoids were calculated by generating the respective quinones through oxidation with an excess of periodate. Oxidation of these substrates by this reagent was analogous to oxidation by tyrosinase with molecular oxygen, although the procedure showed several advantages over the enzymatic method in that oxidation took place almost immediately and quinone stability was favored because no substrate remained. The o-diphenols studied were pyrocatechol, 4-methylcatechol, 4-tert-butylcatechol, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylethylamine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, and caffeic acid; the triphenols studied were pyrogallol, 1,2,4-benzenetriol, 6-hydroxydopa, and 6-hydroxydopamine; and the flavonoids studied were (+)catechin, (-)epicatechin, and quercetin. In addition, the stability of the quinones generated by oxidation of the compounds by [periodate]0/[substrate]0 < 1 was studied. Taking the findings into account, tyrosinase could be measured by following o-quinone formation in rapid kinetic studies using the stopped-flow method. However, measuring o-quinone formation could not be useful for steady-state studies. Therefore, several methods for following tyrosinase activity are proposed, and a kinetic characterization of the enzyme's action on these substrates is made.     

Selective uptake of 2-thiouracil into melanin-producing systems depends on chemical binding to enzymically generated dopaquinone

2-Thiouracil (TU), an antithyroid drug, is receiving growing interest as a specific tumor marker for malignant melanoma, owing to its capability of being selectively accumulated into active melanin-producing tissues. However, up until now, the molecular mechanism of TU uptake by growing melanin has remained largely unknown. In an attempt to fill this gap, we have investigated the effect of TU on the tyrosinase catalyzed oxidation of tyrosine. At a concentration of 0.5 mM, TU was found to totally inhibit melanin formation by tyrosinase catalyzed oxidation of 0.25 mM tyrosine in phosphate buffer at pH 6.8. Polarographical monitoring of oxygen consumption under conditions of complete suppression of melanogenesis revealed a significant tyrosinase activity, with TU acting as a modest non-competitive inhibitor of the enzyme (Ki = 0.6 mM). HPLC and TLC analysis of the tyrosine-tyrosinase reaction in the presence of excess TU showed that the substrate is progressively consumed and a major hitherto unknown product (lambda max = 284 nm), positive to ninhydrin and ferric chloride, is concomitantly formed. This was isolated by repeated gel filtration chromatography of the reaction mixture on Sephadex G-10 and was formulated as the TU-dopa adduct 3,4-dihydroxy-6-(4'-hydroxypyrimidinyl-2'-thio)phenylalanine by spectral analysis. These results suggest that selective TU incorporation in pigmented melanomas and other melanin-producing systems is due to the covalent binding to dopaquinone, produced by tyrosinase catalyzed oxidation of tyrosine.     

盐生植物盐地碱蓬酪氨酸酶的酶学特性

酪氨酸酶是植物甜菜素生物合成的限速酶,但是,其酶学特性尚不了解。以黑暗培养3d的盐地碱蓬幼苗为材料,采用NaF抽提、饱和硫酸铵沉淀法提取盐地碱蓬中的酪氨酸酶,以研究其酶学特性。结果表明,酪氨酸酶氧化活性的最适温度为35℃,最适pH值为6.6,最适条件下Km=1.09mmol·L-1,Vmax=71.43μmol·g-1（FW）·min-1;酪氨酸酶羟化活性的最适温度为40℃,最适pH值为6.6,最适条件下Km=3.16mmol·L-1,Vmax=0.645μmol·g-1（FW）·min-1。Na2S2O3是盐地碱蓬酪氨酸酶的强效抑制剂,0.05mol·L-1Na2S2O3几乎完全抑制酪氨酸酶氧化及羟化活性。而0.01mmol·L-1的Cu2＋可以显著激活酪氨酸酶的氧化及羟化活性,分别为对照的126%和128.2%。这些结果表明盐地碱蓬中酪氨酸酶的羟化活性是影响甜菜红素合成速率的关键,也为深入研究盐地碱蓬酪氨酸酶在甜菜素合成中的作用及其与环境之间的关系奠定了基础。     

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.     

Hydroxylated serotonin and dopamine as substrates and inhibitors for human cytosolic SULT1A3

Sulfation as catalyzed by the cytosolic sulfotransferases (SULTs) is known to play an important role in the regulation and homeostasis of monoamine neurotransmitters. The current study was designed to examine the occurrence of the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine by human cytosolic SULTs and to investigate the inhibitory effects of these hydroxylated derivatives on the sulfation of their unhydroxylated counterparts, serotonin and dopamine. A systematic study using 11 known human cytosolic SULTs revealed SULT1A3 as the responsible enzyme for the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine. The pH-dependence and kinetic constants of SULT1A3 with 7-hydroxyserotonin or 6-hydroxydopamine as substrate were determined. The inhibitory effects of 7-hydroxyserotonin and 6-hydroxydopamine on the sulfation of serotonin and dopamine were evaluated. Kinetic analyses indicated that the mechanism underlying the inhibition by these hydroxylated monoamine derivatives is of a competitive-type. Metabolic labeling experiments showed the generation and release of [³⁵S]sulfated 7-hydroxyserotonin and [³⁵S]sulfated 6-hydroxydopamine when SK-N-MC human neuroblastoma cells were labeled with [³⁵S]sulfate in the presence of 7-hydroxyserotonin or 6-hydroxydopamine. Upon transfection of the cells with siRNAs targeted at SULT1A3, diminishment of the SULT1A3 protein and concomitantly the sulfating activity toward these hydroxylated monoamines was observed. Taken together, these results indicated clearly the involvement of sulfation in the metabolism of 7-hydroxyserotonin and 6-hydroxydopamine. By serving as substrates for SULT1A3, these hydroxylated monoamines may interfere with the homeostasis of endogenous serotonin and dopamine.     

pH-dependent interconvertible forms of mushroom tyrosinase with different kinetic properties

The lag in cresolase activity and inhibition by excess tyrosine of mushroom tyrosinase which was observed when assayed at pH 6.8 was found to be absent when assayed at pH 5.0. The absence of lag and inhibition by excess tyrosine of tyrosinase at pH 5.0 were brought about only after the enzyme was kept at pH 5.0, at 0-4 degrees C, for 1.5 h. The enzyme kept at pH 5.0 for 1.5-3 h at 0-4 degrees C when brought back to pH 6.8, acquires lag and inhibition by excess tyrosine when its activity was measured at pH 6.8. The pH-dependent changes in the kinetic properties of the mushroom tyrosinase are similar to the pH-dependent changes in the kinetic properties of tyrosinase from B-16 murine melanoma and human skin, and thus appear to be a general property of tyrosinase from diverse sources.     

Kinetic study of the transient phase of a chemical reaction system coupled to an enzymatically catalyzed step. Application to the oxidation of epinine by tyrosinase

The present work deals with epinine oxidation by mushroom tyrosinase and sodium metaperiodate. Intermediates produced within short reaction times were characterized by repetitive scanning spectrophotometry and the stoichiometry of the appearance of the respective aminochrome was established. The oxidation pathway from epinine to aminochrome had the following steps: epinine----o-quinone-H+----o-quinone----leukoaminochrome----aminoc hrome. The stoichiometry for the conversion of o-quinone-H+ into the aminochrome of epinine followed the equation: 2 o-quinone-H+----epinine+aminochrome. A transient phase kinetic study has been developed for the system of chemical reactions coupled to an enzymatically catalyzed step, these taking place when epinine is oxidized by mushroom tyrosinase. Rate constants for the implied chemical steps at different temperature and pH values were calculated from analysis of the progress curves of aminochrome accumulation with time. The thermodynamic activation parameters of the chemical steps were also calculated.