Effects of anions, Ca2+, Mg2+, and aliphatic alcohols on the reaction of hemocyanin with cyanide

The reaction between Carcinus maenas hemocyanin and cyanide has been used for probing protein conformation in the presence of perturbants such as various anions, cations (Ca2+, Mg2+), and aliphatic alcohols. The kinetic parameters of the reaction are strongly affected by these agents, suggesting that the induced conformational modifications change the reactivity of the active site toward exogenous ligands. Different patterns are observed according to the perturbant used. As indicated by the mathematical treatment of the kinetic curves the affinity of the active site for CN- and O2 is affected much more than the rate constant of copper removal.     

Kinetic evaluation of catalase and peroxygenase activities of tyrosinase

Tyrosinase is a copper monooxygenase containing a coupled dinuclear copper active site (type-3 copper), which catalyzes oxygenation of phenols (phenolase activity) as well as dehydrogenation of catechols (catecholase activity) using O(2) as the oxidant. In this study, catalase activity (conversion of H(2)O(2) to (1/2)O(2) and H(2)O) and peroxygenase activity (H(2)O(2)-dependent oxygenation of substrates) of mushroom tyrosinase have been examined kinetically by using amperometric O(2) and H(2)O(2) sensors. The catalase activity has been examined by monitoring the initial rate of O(2) production from H(2)O(2) in the presence of a catalytic amount of tyrosinase in 0.1 M phosphate buffer (pH 7.0) at 25 degrees C under initially anaerobic conditions. It has been found that the catalase activity of mushroom tyrosinase is three-order of magnitude greater than that of mollusk hemocyanin. The higher catalase activity of tyrosinase could be attributed to easier accessibility of H(2)O(2) to the dinuclear copper site of tyrosinase. Mushroom tyrosinase has also been demonstrated for the first time to catalyze oxygenation reaction of phenols with H(2)O(2) (peroxygenase activity). The reaction has been investigated kinetically by monitoring the H(2)O(2) consumption rate in 0.5 M borate buffer (pH 7.0) under aerobic conditions. Similarity of the substituent effects of a series of p-substituted phenols in the peroxygenase reaction with H(2)O(2) to those in the phenolase reaction with O(2) as well as the absence of kinetic deuterium isotope effect with a perdeuterated substrate (p-Cl-C(6)D(4)OH vs p-Cl-C(6)H(4)OH) clearly demonstrated that the oxygenation mechanisms of phenols in both systems are the same, that is, the electrophilic aromatic substitution reaction by a (micro-eta(2):eta(2)-peroxo)dicopper(II) intermediate of oxy-tyrosinase.     

Activation mechanism of melB tyrosinase from Aspergillus oryzae by acidic treatment

The pro form of recombinant tyrosinase from Aspergillus oryzae (melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O₂-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh in J. Am. Chem. Soc. 125:13034–13035, 2003) and activated hemocyanin with urea (Morioka et al. in J. Am. Chem. Soc. 128:6788–6789, 2006).     

Interactions of manganese(II) and small ligands with Busycon canaliculatum hemocyanin

Cyanide (5 X 10(-3) M) and thioacetamide (5 X 10(-3) M) increase the P50 values (P02 required for 50% oxygenation) of hemocyanin by 100%, respectively. Using an ion-exchange method involving 14CN-, we have found that cyanide forms a 1:1 complex with hemocyanin in the concentration range examined: Kf = 2.3 X Mw M-1 at room temperature, where Kf is association constant and Mw is molecular weight of hemocyanin. This strong binding of cyanide to hemocyanin is to be expected from the effect of this ion on the oxygenation of hemocyanin. The effects of manganese(II) ion and fluoride on the oxygenation of hemocyanin are found to be weak. The nmr measurements, however, suggest that manganese(II) ion does have some interactions with the active site of hemocyanin.     

A bridged Mo(VI) complex containing six and five coordinate Mo: Synthesis, 95Mo N.m.r. spectroscopy and X-ray crystal structure of Mo2O5(C17H17N2O)2

The kinetics of the reaction between Carcinus maenas hemocyanin and cyanide has been studied at various KCN concentrations and a different temperatures (21° and 4°C) by following the decrease of the copper-peroxide absorption band, centered at 337 nm, of the copper still bound to the protein and the intrinsic fluorescence changes as functions of time. In all conditions used, the absorption band completely disappears and KCN concentration affects only the rate of the process. The reaction is kinetically homogeneous indicating no site-site interaction. The apparent rate constant increases with the square of cyanide concentration and the inverse of O₂ concentration. The copper still bound decreases at a rate slower than the 337 nm absorption and the process is not kinetically homogeneous. The fluorescence of the protein increases after an induction period showing an inflection point at about 50% of the total effect. A kinetic model has been proposed on the assumption that the two metal ions are removed sequentially from the active site. The experimental data are in agreement with the theoretical equations derived from the model. The equilibrium constants for the formation of the complex between the first and the second copper ion with cyanide and the rate constants of their decomposition have been calculated. The rate-limiting process for the removal of the second copper ion is the formation of the complex with cyanide.     

Displacement of O2 and CO by cyanide from the active site of helix pomatia β-hemocyanin.: Formation of a mixed-liganded species

Addition of KCN to Helix pomatia β-hemocyanin fully saturated with either O₂ or CO results in a decrease of the spectroscopic properties of the protein (absorbance at 340 nm and luminescence at 550 nm) due to the displacement of the gaseous ligands (O₂ or CO) from the active site. The anionic form of cyanide (CN^?) is supposed to bind to the active site; its intrinsic affinity for the protein, as calculated from independent O₂ and CO displacement experiments, is between 2 and 6 × 10⁶M^?1. The replacement of O₂ or CO shows some differences which may be correlated with the different modes of binding at the active site. Thus, while displacement of oxygen by cyanide is hyperbolic, addition of cyanide to carbonylated hemocyanin shows a lag phase. This finding suggests the formation of a mixed liganded complex at the active site. The simultaneous presence of CO and CN^? at the active site of hemocyanin is also supported by the experiment in which addition of small amounts of KCN to hemocyanin partially saturated with O₂ and CO gives rise to an increase of emission intensity and a concomitant decrease of the O₂ absorption band. The mixed-liganded species displays luminescence properties similar to those of CO-saturated hemocyanin, and the formation of the complex is reversible on dialysis or oxygenation.     

Effect of thiohydroxyl compounds on tyrosinase: inactivation and reactivation study

An unusual thioether bridge (Cys-His) has been detected at the active site of mushroom tyrosinase, and the effects of thiohydroxyl compounds such as dithiothreitol (DTT) and beta-mercaptoethanol (beta-ME) on Cu2+ at the active site have been elucidated. Treatment with DTT and beta-ME on mushroom tyrosinase completely inactivated 3,4-dihydroxyphenylalanine oxidase activity in a dose-dependent manner. Sequential kinetic studies revealed that DTT and beta-ME caused different mixed-type inhibition mechanisms: the slope-parabolic competitive inhibition (Ki = 0.143 mM) by DTT and slope-hyperbolic noncompetitive inhibition (Ki = 0.0128 mM) by beta-ME, respectively. Kinetic Scatchard analysis consistently showed that mushroom tyrosinase had multiple binding sites for DTT and beta-ME with different affinities. Reactivation study of inactivated enzyme by addition of Cu2+ confirmed that DTT and beta-ME directly bound with Cu2+ at the active site. Our results may provide useful information regarding interactions of tyrosinase inhibitor for designing an effective whitening agent targeted to the tyrosinase active site.     

Inhibition kinetics of mushroom tyrosinase by copper-chelating ammonium tetrathiomolybdate

With a strategy of chelating coppers at tyrosinase active site to detect an effective inhibitor, several copper-specific chelators were applied in this study. Ammonium tetrathiomolybdate (ATTM) among them, known as a drug for treating Wilson's disease, turned out to be a significant tyrosinase inhibitor. Treatment with ATTM on mushroom tyrosinase completely inactivated enzyme activity in a dose-dependent manner. Progress-of-substrate reaction kinetics using the two-step kinetic pathway and dilution of the ATTM revealed that ATTM is a tight-binding inhibitor and high dose of ATTM irreversibly inactivated tyrosinase. Progress-of-substrate reaction kinetics and activity restoration with a dilution of the ATTM indicated that the copper-chelating ATTM may bind slowly but reversibly to the active site without competition with substrate, and the enzyme-ATTM complex subsequently undergoes reversible conformational change, leading to complete inactivation of the tyrosinase activity. Thus, inhibition by ATTM on tyrosinase could be categorized as complexing type of inhibition with a slow and reversible binding. Detailed analysis of inhibition kinetics provided IC50 at the steady-state and inhibitor binding constant (K(I)) for ATTM as 1.0+/-0.2 microM and 10.65 microM, respectively. Our results may provide useful information regarding effective inhibitor of tyrosinase as whitening agents in the cosmetic industry.     

Tyrosinase activity and hemocyanin in the hemolymph of the slipper lobster <Emphasis Type="Italic">Scyllarides latus</Emphasis>

The respiratory protein hemocyanin is present in molluscans and in some species of arthropods, and its dioxygen binding site strongly resembles that of the monophenol-hydroxylating and catechol-quinonising enzyme tyrosinase. Moreover, some hemocyanins show a certain extent of tyrosinase activity, so a common ancestry between the two proteins has been suggested. However, in the case purified hemocyanin of Scyllarides latus any attempts to evoke tyrosinase activity failed. A distinct tyrosinase has been purified to homogeneity from the hemolymph, and kinetically characterised. The purified tyrosinase showed both monophenolase and diphenolase enzyme activity and therefore it could be well defined as a true tyrosinase. This finding suggests that in the case of the studied crustacean the evolutionary functional divergence between dioxygen transport and oxidation of phenolics has already reached its completeness.     

Similar enzyme activation and catalysis in hemocyanins and tyrosinases

This review presents the common features and differences of the type 3 copper proteins with respect to their structure and function. In spite of these differences a common mechanism of activation and catalysis seems to have been preserved throughout evolution. In all cases the inactive proenzymes such as tyrosinase and catecholoxidase are activated by removal of an amino acid blocking the entrance channel to the active site. No other modification at the active site seems to be necessary to enable catalytic activity. Hemocyanins, the oxygen carriers in many invertebrates, also behave as silent inactive enzymes and can be activated in the same way. The molecular basis of the catalytic process is presented based on recent crystal structures of tyrosinase and hemocyanin. Minor conformational differences at the active site seem to decide about whether the active site is only able to oxidize diphenols as in catecholoxidase or if it is also able to o-hydroxylate monophenols as in tyrosinase.     

Photoinactivation of agaricus bisporus tyrosinase: modification of the binuclear copper site

Irradiation of Agaricus bisporus tyrosinase in the presence of citrate at pH 5.6 with 300-420-nm light results in a loss of both catecholase activity and cresolase activity. The light-sensitive species appears to be an enzyme-citrate complex, most likely involving coordination of citrate to the active site copper. One copper ion from each binuclear active site can be removed from the inactivated enzyme, resulting in the formation of a met apo derivative. The electron spin resonance spectrum of met apo tyrosinase resembles that of met apo hemocyanin and half-met Neurospora tyrosinase. It is consistent with a distorted square-planar geometry around the copper and with either nitrogen or nitrogen and oxygen ligands. Amino acid analysis indicates that four histidines on the heavy subunit are destroyed during the inactivation process. Some or all of these histidines may serve as ligands to the copper ion which becomes labile after inactivation. Photoinactivation results in decarboxylation of citrate and does not require the presence of oxygen. The reaction may involve generation of a free radical from the citrate which then attacks nearby histidine residues.     

The effect of thiobarbituric acid on tyrosinase: inhibition kinetics and computational simulation

Tyrosinase plays various roles in organisms and much research has focused on the regulation of tyrosinase activity. We studied the inhibitory effect of thiobarbituric acid (TBA) on tyrosinase. Our kinetic study showed that TBA inhibited tyrosinase in a reversible noncompetitive manner (K(i) 5 14.0 ± 8.5 mM and IC?? 5 8.0 ± 1.0 mM). Intrinsic and ANS-binding fluorescences studies were also performed to gain more information regarding the binding mechanism. The results showed that no tertiary structural changes were obviously observed. For further insight, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and TBA. The docking simulation was successful with significant scores (binding energy for AutoDock4: -5.52 kcal/mol) and suggested that TBA was located in the active site. The 11 ns molecular dynamics simulation convinced that the four HIS residues (residue numbers: 57, 90, 250, and 282) were commonly responsible for the interaction with TBA. Our results provide a new inhibition strategy that works using an antioxidant rather than targeting the copper ions within the tyrosinase active site.     

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.     

Phenoloxidase activity of intact and chemically modified functional unit RvH1: a from molluscan Rapana venosa hemocyanin

o-Diphenol oxidase activities (o-diPO) of chemically modified functional unit RvH1-a of molluscan hemocyanin Rapana venosa were studied using L-Dopa and dopamine as substrates. With L-Dopa as substrate the native FU RvH1-a did not show any o-diPO activity. Therefore the native FU RvH1-a was converted to enzymatic active form, after treatment with SDS, trypsin, urea and different values of pH when its o-diPO activity was studied. The highest artificial induction of o-diPO activity was observed after incubation of FU with 3.0mM SDS, and RvH1-a shows both, dopamine (K(M)=6.53mM, k(cat)/K(M)=1.29) and L-Dopa (K(M)=2.0mM, k(cat)/K(M)=2.1) activity due to a more open active site of the enzyme and better access of the substrates. It was determined that the K(M) value of SDS-activated RvH1-a against dopamine is higher compared to those of hemocyanins from Helix vulgaris, Helix pomatia and native tyrosinase from Ipomoea batatas but much lower than that from Illex argentinus (ST94) tyrosinase and arthropodan hemocyanin from Carcinus aestuarii. The Km value of SDS-activated RvH1-a against L-Dopa is higher than those of hemocyanins from H. vulgaris and Cancer magister, but lower than that of the tyrosinase from Streptomyces albus.     

The reaction of Octopus vulgaris hemocyanin with exogenous ligands: proposal of an allosteric model for the binding of cyanide and thiourea to the 11 S subunit

Octopus vulgaris hemocyanin in 11 S aggregation state binds oxygen following a noncooperative oxygen saturation curve with Hill coefficient n = 1. Under the same conditions the equilibrium and kinetics of the reaction with cyanide and other ligands are indicative of an anticooperative behavior displaying different characteristics for the different ligands. The data are consistent with an induced-fit type allosteric model which assumes for the 11 S subunit of O. vulgaris hemocyanin an annular structure made up by five identical domains each containing one binding site whose reactivity is near-neighbor regulated.     

Conformational stabilization at the active site of molluskan (Rapana thomasiana) hemocyanin by a cysteine-histidine thioether bridge A study by mass spectrometry and molecular modeling

In some type-3 copper proteins (molluskan hemocyanin, catechol oxidase and fungal tyrosinase) one of the histidine residues, liganding the Cu(A) atom of the dinuclear copper active site, is covalently linked to a cysteine residue by a thioether bridge. The purpose of this study was to disclose the function of this bridge. Mass spectral analysis of a peptide, isolated from Rapana thomasiana (gastropodan mollusk) hemocyanin, indicated a stabilization of the peptide structure in the region of the bridge. Molecular modeling of three thioether containing type-3 copper proteins using the dead-end elimination method showed that the concerned histidine would be very flexible if not linked to the cysteine. Also, the side chain orientation of the histidine is rather exceptional, as evidenced by statistical data from the protein databank. It is suggested that the role of the bridge is to fix the histidine in an orientation that is optimal for coordination of the Cu(A) atom.     

Structural basis and mechanism of the inhibition of the type-3 copper protein tyrosinase from Streptomyces antibioticus by halide ions

The inhibition of the type-3 copper enzyme tyrosinase by halide ions was studied by kinetic and paramagnetic (1)H NMR methods. All halides are inhibitors in the conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) with apparent inhibition constants that follow the order I(-) < F(-) < Cl(-) < Br(-) at pH 6.80. The results show that the inhibition arises from the interaction of halide with both the oxidized (affinity F(-) > Cl(-) > Br(-) > I(-)) and reduced (affinity I(-) > Br(-) > Cl(-) > F(-)) enzyme. The paramagnetic (1)H NMR of the oxidized enzyme complexed with the halides is consistent with a direct interaction of halide with the type-3 site and shows that the (Cu-His(3))(2) coordination occurs in all halide-bound species. It is surmised that halides bridge both of the copper ions in the active site. Fluoride and chloride are shown to bind only to the low pH form of oxidized tyrosinase, explaining the strong pH dependence of the inhibition by these ions. We further show that p-toluic acid and the bidentate transition state analogue, Kojic acid, displace chloride from the oxidized active site, whereas the monodentate substrate analogue, p-nitrophenol, forms a ternary complex with the enzyme and the chloride ion. On the basis of the experimental results, a model is formulated for the inhibitor action and for the reaction of diphenols with the oxidized enzyme.     

Switch between tyrosinase and catecholoxidase activity of scorpion hemocyanin by allosteric effectors

Phenoloxidases and hemocyanins have similar type 3 copper centers although they perform different functions. Hemocyanins are oxygen carriers, while phenoloxidases (tyrosinase/catecholoxidase) catalyze the initial step in melanin synthesis. Tyrosinases catalyze two subsequent reactions, whereas catecholoxidases catalyze only the second one. Recent results indicate that hemocyanins can also function as phenoloxidases and here we show for the first time that hemocyanin can be converted to phenoloxidase. Furthermore, its substrate specificity can be switched between catecholoxidase and tyrosinase activity depending on effectors such as hydroxymethyl-aminomethan (Tris) and Mg(2+)-ions. This demonstrates that substrate specificity is not caused by a chemical modification of the active site.     

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.     

Inhibition of tyrosinase by green tea components

The pigment melanin in human skin is a major defense mechanism against ultraviolet light of the sun, but darkened skin color, which is the result of increased and redistributed epidermal melanin, could be a serious aesthetic problem. Epidemiologically, it is well known that the consumption of green tea may help prevent cancers in humans and also reduce several free radicals including peroxynitrite. In the present study, to assess the efficacy of the inhibition of mushroom tyrosinase (monophenol monooxygenase EC 1.14.18.1), ten kinds of Korean traditional teas were screened for their tyrosinase inhibitory activity. Green tea was the strongest inhibitor, and the major active constituents in the tea are (-)-epicatechin 3-O-gallate (ECG), (-)-gallocatechin 3-O-gallate (GCG), and (-)-epigallocatechin 3-O-gallate (EGCG). All are catechins with gallic acid group as an active site. The kinetic analysis for inhibition of tyrosinase revealed a competitive nature of GCG with this enzyme for the L-tyrosine binding at the active site of tyrosinase.