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Several novel quinazoline derivatives bearing phenolic hydroxyl moiety (2–7) have been produced with good yields and screened for biological activities. All the title compounds were characterized using spectroscopic techniques such as 1H NMR, 13C NMR, FTIR, and HRMS. Then, the anti-browning effects of synthesized quinazoline derivatives were investigated in vitro. The IC50 values for molecules 2–7 were calculated as 0.085, 1.145, 0.106, 6.86, 0.52, 7.07 µM, respectively. Ki constants, which are inhibitory-enzyme binding constants, were calculated by using Lineweaver–Burk graphs as 0.16 ± 0.0620, 0.906 ± 0.3029, 0.055 ± 0.0171, 9.363 ± 2.5809, 0.773 ± 0.3204, 7.863 ± 1.9107 µM, respectively. In computer-aided analysis, to gain insights electrochemical properties, synthesized compounds were analysed theoretically by density functional theory. Molecular docking studies and MD simulations were performed to identify possible inhibitor-enzyme binding interactions. According to obtained results, all the compounds formed hydrogen bonds with Asn 112 and Asn 414, and showed π-cation interaction with Phe2 70, gatekeeper residue in target protein. Supporting the nm level inhibition, MD simulations indicate that protein-inhibitor complex maintain the stability and have high number of hydrogen bond formation during the simulation. [ABSTRACT FROM AUTHOR]

The trend of modelling native enzymes is increasing day by day with the motive of mechanistic elucidation of enzyme-catalyzed reactions and the efficiency of catalysts. Most of the metalloenzymes are capable to activate molecular oxygen due to the presence of metal ions. Among these, copper-based metalloenzyme catechol oxidase has an attractive feature of its oxidizing ability to generate ortho-quinones from catechol. Moreover, the conversion of catechol to ortho-quinones is an important tool for the determination of hormonally active catecholamines such as adrenaline, noradrenaline, and dopa. Nevertheless, the use of native enzymes is highly expensive and the employment of biomimetic models can be the best alternative. This review summarizes some of the widely used modelling methods and their successful applications. Additionally, we also briefly elucidated the structure–activity relationship, kinetic studies of the catalytic oxidation of the substrate, and different external factors influencing the catalytic cycle such as pH, temperature, etc. [ABSTRACT FROM AUTHOR]

Polyphenol oxidases (PPOs) comprise tyrosinases (TYRs) and catechol oxidases (COs), which catalyse the initial reactions in the biosynthesis of melanin. TYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas COs react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono- versus diphenolase activity in the type-III copper enzyme jrPPO1. [ABSTRACT FROM AUTHOR]

Copper(II) complexes of five peptide ligands containing at least three histidine residues have been tested as catalysts in catechol oxidation and superoxide dismutation. All systems exhibit considerable catechol oxidase-like activity, and the Michaelis-Menten enzyme kinetic model is applicable in all cases. Beside the Michaelis-Menten parameters, the effects of pH, catalyst and dioxygen concentration on the reaction rates are also reported. Considering the rather different sequences, the observed oxidase activity seems to be a general behavior of copper(II) complexes with multihistidine peptides. Interestingly, in all cases {Nim/2Nim,2N−} coordinated complexes are the pre-active species, the bound amide nitrogens were proposed to be an acid/base site for facilitating substrate binding. The studied copper(II)-peptide complexes are also able to effectively dismutate superoxide radical in the neutral pH range. [ABSTRACT FROM AUTHOR]

Abstract: This paper presents the synthesis and characterization of Cu(II) and Fe(III) complexes of a newly synthesized Mannich base N-[4-hydroxy-3-[[4-(pyridin-2-yl)piperazin-1-yl]methyl]phenyl]acetamide. The spectro-analytical techniques employed include 1H, 13C NMR, IR, UV spectroscopy, conductivity measurements, and elemental analysis. Both mononuclear and dinuclear metal complexes were encountered in the study. Utilizing 3,5-di-tert-butyl catechol (3,5-DTBC) as the substrate, catecholase activity of all four complexes was evaluated with the detailed kinetic studies reported. The nature of the bonding of the thiocyanate is discussed based on the observation of νCN (2150-2050 cm−1). All the metal complexes are catalytically active with turn over numbers ranging from 9.47 to 31.86 h−1 in DMF.Graphical abstract: [ABSTRACT FROM AUTHOR]

The authors report on a nonenzymatic catechol sensor that is based on the immobilization of ferrocene (Fc) on graphene oxide (GO). A glassy carbon electrode (GCE) was modified with GO which then was silanized with (3-aminopropyl)trimethoxysilane. Ferrocenecarboxaldehyde was then immobilized on GO via formation of a Schiff base. The immobilization process was monitored stepwise by using FTIR spectroscopy, X-ray diffraction, cyclic voltammetry (CV) and electrochemical impedance spectroscopy. Investigation of the modified electrode by CV revealed a pair of well-defined redox peaks with anodic and cathodic peak potentials at +0.380 and +0.277 V, corresponding to the Fc/Fc redox couple. The Fc modified electrode exhibits excellent electrocatalytic activity towards the oxidation of catechol at a typical working voltage of +0.45 V (vs. Ag/AgCl). The response is linear in the 3 to 112 μM catechol concentration range, the detection limit is 1.1 μM, and the sensitivity is 1184.3 μA·mM·cm. The sensor is stable, reproducible and reasonably selective. It was successfully applied to the determination of catechol in spiked tap water and lake water samples. [ABSTRACT FROM AUTHOR]

A series of N-phenylthiourea derivatives was built starting from the X-ray structure in the molecular mechanics framework and the interaction profile in the complex with the catechol oxidase was traced using molecular dynamics simulation. The results showed that the geometry and interactions between ligand and receptor were highly related to the position of the substituted side chains of phenyl moiety. At the end of molecular dynamics run, a concentrated multicenter hydrogen bond was created between the substituted ligand and receptor. The conformation of the ligand itself were also restricted in the receptor pocket. Furthermore, the simulation time of 50 ns were found to be long enough to explore the relevant conformational space and the stationary behavior of the molecular dynamic could be observed. [ABSTRACT FROM AUTHOR]

An unprecedented solid of coper(II) complexes, [Cu(dpa)NCS][Cu(dpa)(NCS)](ClO) ( 1) [dpa = 2,2′-dipyridylamine; SCN = thiocyanate], has been synthesized and crystallographically characterized with the aim to study the catecholase activity. The Cu(II) complex mimics the full catalytic cycle of the active site of catechol oxidase enzyme in acetonitrile medium with a turnover number of 4.788×10 h along with the production of semiquinone radical and hydrogen peroxide. In situ generation of Cu(I) species in the catalytic pathway of catechol oxidation was established by electrochemical study and further confirmed by electron paramagnetic resonance (EPR) spectroscopy. [ABSTRACT FROM AUTHOR]

Four new imidazole-based ligands, 4-((1 H-imidazol-4-yl)methyl)-2-phenyl-4,5-dihydrooxyzole (L 1), 4-((1 H-imidazol-4-yl)methyl)-2-( tert-butyl)-4,5-dihydrooxyzole (L 2), 4-((1 H-imidazol-4-yl)methyl)-2-methyl-4,5-dihydrooxyzole (L 3), and N-(2,2-dimethylpropylidene)-2-(1-trityl-1 H-imidazol-4-yl-)ethyl amine ( L 1), have been synthesized. The corresponding copper(I) complexes [Cu(I)( L 1)(CHCN)]PF (Cu L 1), [Cu(I)( L 2)(CHCN)]PF (Cu L 2), [Cu(I)( L 3)(CHCN)]PF (Cu L 3), [Cu(I)( L 1)(CHCN)]PF (Cu L 1) as well as the Cu(I) complex derived from the known ligand bis(1-methylimidazol-2-yl)methane (BIMZ), [Cu(I)( BIMZ)(CHCN)]PF (Cu BIMZ), are screened as catalysts for the oxidation of 3,5-di- tert-butylcatechol (3,5-DTBC-H) to 3,5-di- tert-butylquinone (3,5-DTBQ). The primary reaction product of these oxidations is 3,5-di- tert-butylsemiquinone (3,5-DTBSQ) which slowly converts to 3,5-DTBQ. Saturation kinetic studies reveal a trend of catalytic activity in the order Cu L 3 ≈ Cu L 1 > Cu BIMZ > Cu L 2 > Cu L 1. Additionally, the catalytic activity of the copper(I) complexes towards the oxygenation of monophenols is investigated. As substrates 2,4-di- tert-butylphenol (2,4-DTBP-H), 3- tert-butylphenol (3-TBP-H), 4-methoxyphenol (4-MeOP-H), N-acetyl- l-tyrosine ethyl ester monohydrate (NATEE) and 8-hydroxyquinoline are employed. The oxygenation products are identified and characterized with the help of UV/Vis and NMR spectroscopy, mass spectrometry, and fluorescence measurements. Whereas the copper complexes with ligands containing combinations of imidazole and imine functions or two imidazole units (Cu L 1 and Cu BIMZ) are found to exhibit catalytic tyrosinase activity, the systems with ligands containing oxazoline just mediate a stoichiometric conversion. Correlations between the structures of the complexes and their reactivities are discussed. [ABSTRACT FROM AUTHOR]

Catechol oxidases (EC 1.10.3.1) catalyse the oxidation of o-diphenols to their corresponding o-quinones. These oxidases contain two copper ions (CuA and CuB) within the so-called coupled type 3 copper site as found in tyrosinases (EC 1.14.18.1) and haemocyanins. The crystal structures of a limited number of bacterial and fungal tyrosinases and plant catechol oxidases have been solved. In this study, we present the first crystal structure of a fungal catechol oxidase from Aspergillus oryzae (AoCO4) at 2.5-Å resolution. AoCO4 belongs to the newly discovered family of short-tyrosinases, which are distinct from other tyrosinases and catechol oxidases because of their lack of the conserved C-terminal domain and differences in the histidine pattern for CuA. The sequence identity of AoCO4 with other structurally known enzymes is low (less than 30 %), and the crystal structure of AoCO4 diverges from that of enzymes belonging to the conventional tyrosinase family in several ways, particularly around the central α-helical core region. A diatomic oxygen moiety was identified as a bridging molecule between the two copper ions CuA and CuB separated by a distance of 4.2–4.3 Å. The UV/vis absorption spectrum of AoCO4 exhibits a distinct maximum of absorbance at 350 nm, which has been reported to be typical of the oxy form of type 3 copper enzymes. [ABSTRACT FROM AUTHOR]

Catechol oxidases (EC 1.10.3.1) catalyse the oxidation of o-diphenols to their corresponding o-quinones. These oxidases contain two copper ions (CuA and CuB) within the so-called coupled type 3 copper site as found in tyrosinases (EC 1.14.18.1) and haemocyanins. The crystal structures of a limited number of bacterial and fungal tyrosinases and plant catechol oxidases have been solved. In this study, we present the first crystal structure of a fungal catechol oxidase from Aspergillus oryzae (AoCO4) at 2.5-Å resolution. AoCO4 belongs to the newly discovered family of short-tyrosinases, which are distinct from other tyrosinases and catechol oxidases because of their lack of the conserved C-terminal domain and differences in the histidine pattern for CuA. The sequence identity of AoCO4 with other structurally known enzymes is low (less than 30 %), and the crystal structure of AoCO4 diverges from that of enzymes belonging to the conventional tyrosinase family in several ways, particularly around the central α-helical core region. A diatomic oxygen moiety was identified as a bridging molecule between the two copper ions CuA and CuB separated by a distance of 4.2–4.3 Å. The UV/vis absorption spectrum of AoCO4 exhibits a distinct maximum of absorbance at 350 nm, which has been reported to be typical of the oxy form of type 3 copper enzymes. [ABSTRACT FROM AUTHOR]

A series of seven heterocyclic compounds based on pyridazinone and thiopyridazinone moieties: 5-(2-chlorobenzyl)-6-methylpyridazin-3-one L1; 5-[(2-chlorobenzyl)hydroxyl)methyl]6-methylpyridazin-3-one L2; 5-(2-chlorobenzyl)-2,6-dimethylpyridazin-3-one L3; 5-(2-chlorobenzyl)-2-(hydroxyethyl)-6-methylpyridazin-3-one L4; ethyl-4-(2-chloro-benzyl)-3-methyl-6-oxopyridazin-1(6H)-yl)acetate L5; 5-(2-chlorobenzyl)-2-(hydroxyethyl)-6-methylpyridazin-3-thione L6, and ethyl-4-(2-chloro-benzyl)-3-methyl-6-thioxopyridazin-1(6H)-yl)acetate L7 were tested for the oxidation of catechol to o-quinone for miming microorganism in the O activation for electrophilic non substituted aromatic. The in situ generated Cu(II), Fe(II) and Zn(II) complexes of these ligands ( L1- L7) were examined for such catalytic activities. We found that all these substrates catalyze the oxidation reaction of catechol to o-quinone with the presence of atmospheric dioxygen. The rates of this oxidation depend on two parameters: the nature of the ligand and the nature of ion salts. We found that the combination of L7 [ Cu( CH COO)] leads to the fastest catalytic processes. [ABSTRACT FROM AUTHOR]

The oxidase and dioxygenase reactions of 3,5-di- tert-butylcatechol (DTBC, I) in the presence of V-polyoxometalate were studied. It was found that the addition of a Lewis base quenched the V-polyoxometalate-catalysed catechol dioxygenase reaction and catalysed the oxidase reaction selectively. The existence of V-polyoxometalate accelerates the autoxidation rate of I as demonstrated by the rate measurements. ESR and UV-VIS spectra showed that the Lewis base destroyed the dioxygenation reaction catalyst as formed and restrained its regeneration by suppressing the coordination of catechol radical to vanadium. The by-products of the dioxygenation and oxidation reactions are HO and HO, respectively. [ABSTRACT FROM AUTHOR]

Fruit tree leaf tissues were screened in a search for determination of an alternative source(s) for commercial phenol oxidase (PO) production considering the importance of utilization of green biomass for production of value-added products. Mulberry, pear, sour cherry and apricot leaves were identified as promising PO production sources, due to their comparable enzyme activities with respect to mushroom ( Agaricus bisporus), a well-known PO source. Within the scope of this research, further biochemical characterization was only performed for mulberry ( Morus alba) leaf tissue due to its high PO activity (ca. 19 EU g tissue) and also its known non-toxic and edible nature which are important properties of an enzyme source to be used without detailed purification. In mulberry leaves, presence of three different PO activities, laccase, peroxidase and catechol oxidase of 62-64 kDa molecular weights, were identified. Since simple extraction/concentration steps without fractionation/purification was aimed as PO production process, operational parameters such as optimal temperature, pH and kinetic studies of overall PO activity were investigated using concentrated crude extract. The highest PO activity against 4-methyl catechol was observed at 45°C and pH 7. Michaelis-Menten kinetic parameters, K and V, of PO activity were determined as 6 mM 4-methyl catechol and 2.2 μmol quinone produced min ml, respectively. PO activity of mulberry leaves increased up to late November. Consequently, mulberry leaves seem as a suitable PO source for industrial applications in which a wide range of substrate utilization is necessary. [ABSTRACT FROM AUTHOR]

A homology search against public fungal genome sequences was performed to discover novel secreted tyrosinases. The analyzed proteins could be divided in two groups with different lengths (350–400 and 400–600 residues), suggesting the presence of a new class of secreted enzymes lacking the C-terminal domain. Among them, a sequence from Aspergillus oryzae (408 aa, AoCO4) was selected for production and characterization. AoCO4 was expressed in Trichoderma reesei under the strong cbh1 promoter. Expression of AoCO4 in T. reesei resulted in high yields of extracellular enzyme, corresponding to 1.5 g L−1 production of the enzyme. AoCO4 was purified with a two-step purification procedure, consisting of cation and anion exchange chromatography. The N-terminal analysis of the protein revealed N-terminal processing taking place in the Kex2/furin-type protease cleavage site and removing the first 51 amino acids from the putative N-terminus. AoCO4 activity was tested on various substrates, and the highest activity was found on 4- tert-butylcatechol. Because no activity was detected on L-tyrosine and on l-dopa, AoCO4 was classified as a catechol oxidase. AoCO4 showed the highest activity within an acidic and neutral pH range, having an optimum at pH 5.6. AoCO4 showed good pH stability within a neutral and alkaline pH range and good thermostability up to 60°C. The UV–visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct. [ABSTRACT FROM AUTHOR]

The polyphenol oxidase (PPO) of field bean ( Dolichos lablab) is a tetramer made up of two subunits of mass 29,000 and 31,000 Da. The amino acid sequence of the tryptic peptides showed approximately 90% sequence identity to the d-galactose specific legume lectins. The haemagglutinating activity of a pure and homogenous preparation of PPO measured using human erythrocytes was 1261 HAU mg−1 protein and was inhibited by d-galactose. Purification by galactose-sepharose chromatography also indicated that the PPO and haemagglutinating activities were associated with a single protein. Crude extracts of other legumes did not exhibit PPO activity, yet cross reacted with anti-PPO antibodies. This dual function protein with PPO and haemagglutinating activity is unique to field bean. The two activities are independent of each other occurring at different loci on the protein. These observations further evidence and strengthen the assumption that galactose specific legume lectins have enzymatic function. Both PPO and lectins are proteins that play a vital role in the defense mechanism of plants. The complementarity of these two simultaneous and independent powerful defense mechanisms exhibited by a single protein renders it a candidate gene for the development of inbuilt plant protection. [ABSTRACT FROM AUTHOR]

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 ± 0.2 and 10.1 ± 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and l-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases. [ABSTRACT FROM AUTHOR]

A binuclear copper complex, [Cu2(BPMP)(OAc)2][ClO4]·H2O, has been prepared using the binucleating ligand 2,6-bis[bis(pyridin-2-ylmethylamino)methyl]-4-methylphenol (H-BPMP). The X-ray crystal structure reveals the copper centers to have a five-coordinate square pyramidal geometry, with the acetate ligands bound terminally. The bridging phenolate occupies the apical position of the square-based pyramids and magnetic susceptibility, electron paramagnetic resonance (EPR) and variable-temperature variable-field magnetic circular dichroism (MCD) measurements indicate that the two centers are very weakly antiferromagnetically coupled ( J = −0.6 cm−1). Simulation of the dipole–dipole-coupled EPR spectrum showed that in solution the Cu–O–Cu angle was increased from 126° to 160° and that the internuclear distance was larger than that observed crystallographically. The high-resolution spectroscopic information obtained has been correlated with a detailed ligand-field analysis to gain insight into the electronic structure of the complex. Symmetry arguments have been used to demonstrate that the sign of the MCD is characteristic of the tetragonally elongated environment. The complex also displays catecholase activity ( k cat = 15 ± 1.5 min−1, K M = 6.4 ± 1.8 mM), which is compared with other dicopper catechol oxidase models. [ABSTRACT FROM AUTHOR]

The mechanism for the oxidation of catechol by catechol oxidase has been studied using B3LYP hybrid density functional theory. On the basis of the X-ray structure of the enzyme, the molecular system investigated includes the first-shell protein ligands of the two metal centers as well as the second-shell ligand Cys92. The cycle starts out with the oxidized, open-shell singlet complex with oxidation states Cu2(II,II) with a μ-η2:η2 bridging peroxide, as suggested experimentally, which is obtained from the oxidation of Cu2(I,I) by dioxygen. The substrate of each half-reaction is a catechol molecule approaching the dicopper complex: the first half-reaction involves Cu(I) oxidation by peroxide and the second one Cu(II) reduction. The quantitative potential energy profile of the reaction is discussed in connection with experimental data. Since no protons leave or enter the active site during the catalytic cycle, no external base is required. Unlike the previous density functional theory study, the dicopper complex has a charge of +2. [ABSTRACT FROM AUTHOR]

The blue coloured plasma protein ceruloplasmin binds up to 95% of circulating copper, and has several possible functions. It has been proposed to function in copper transport, oxidation of organic amines, iron(II) oxidation and the regulation of cellular iron levels, and catechols, radical scavenging and other antioxidant processes. This account will consider the relative importance of these multiple functions in terms of the physiological roles of ceruloplasmin. [ABSTRACT FROM AUTHOR]

A mechanism for the oxidation of 3,5-di- tert-butylcatechol (dtbc) with dioxygen to the corresponding quinone (dtbq), catalyzed by bispidine-dicopper complexes (bispidines are various mono- and dinucleating derivatives of 3,7-diazabicyclo[3.3.1]nonane with bis-tertiary-amine–bispyridyl or bis-tertiary-amine–trispyridyl donor sets), is proposed on the basis of (1) the stoichiometry of the reaction as well as the stabilities and structures [X-ray, density functional theory (B3LYP, TZV)] of the bispidine-dicopper(II)–3,4,5,6-tetrachlorcatechol intermediates, (2) formation kinetics and structures (molecular mechanics, MOMEC) of the end-on peroxo–dicopper(II) complexes and (3) kinetics of the stoichiometric (anaerobic) and catalytic (aerobic) copper-complex-assisted oxidation of dtbc. This involves (1) the oxidation of the dicopper(I) complexes with dioxygen to the corresponding end-on peroxo–dicopper(II) complexes, (2) coordination of dtbc as a bridging ligand upon liberation of H2O2 and (3) intramolecular electron transfer to produce dtbq, which is liberated, and the dicopper(I) catalyst. Although the bispidine complexes have reactivities comparable to those of recently published catalysts with macrocyclic ligands, which seem to reproduce the enzyme-catalyzed process in various reaction sequences, a strikingly different oxidation mechanism is derived from the bispidine–dicopper-catalyzed reaction. [ABSTRACT FROM AUTHOR]

New tetradentate bis-benzimidazole ligands have been synthesized and utilized to prepare copper (II) complexes. Some of these copper (II) complexes have been characterized structurally. The copper (II) in these complexes is found to possess varying geometries. A distorted octahedral geometry is found with a highly unsymmetrical bidentate nitrate group. An unusual polymeric one-dimensional structure is observed where copper (II) is in a distorted square pyramidal geometry with a monodentate nitrate ion, having long Cu--O bond, while a distorted triagonal bipyramidal geometry is found with two carbonyl O atoms and a Cl atom in the equatorial plane, and two benzimidazole imine N atoms occupy the axial position. These compounds are found to activate the cumylperoxyl group, and this has been utilized in the facile oxidation of aromatic alcohols to aldehydes, where they act as catalysts with large turnovers. The yields of the respective products vary from 32 to 65%. The role of molecular oxygen has been studied and an attempt has been made to identify the "active copper species". Activation of molecular oxygen has also been observed and has been used for oxidative dealkylation of a hindered phenol, producing di-butyl quinones with yields of 20-25% and 10-12 fold catalytic turnover. Dihydroxybenzenes and substituted catechols are also readily oxidized to the corresponding quinones, in oxygen-saturated solvents. Yields of 84% have been observed with 34-fold catalyst turnover, with di-t-butylcatechol. The activity of these copper (II) -- bis-benzmidazolediamide compounds is reminiscent of the functioning of copper centres in galactose oxidase, tyrosinase and catechol oxidase. [ABSTRACT FROM AUTHOR]

The monohydroxo-bridged dicopper(II) complex (1), its reduced dicopper(I) analogue (2) and the trans-μ-1,2-peroxo-dicopper(II) adduct (3) with the macrocyclic N-donor ligand [22]py4pz (9,22-bis(pyridin-2′-ylmethyl)-1,4,9,14,17,22,27,28,29,30- decaazapentacyclo -[22.2.114,7.111,14.117,20]triacontane-5,7(28),11(29),12,18,20(30), 24(27),25-octaene), have been prepared and characterized, including a 3D structure of 1 and 2. These compounds represent models of the three states of the catechol oxidase active site: met, deoxy (reduced) and oxy. The dicopper(II) complex 1 catalyzes the oxidation of catechol model substrates in aerobic conditions, while in the absence of dioxygen a stoichiometric oxidation takes place, leading to the formation of quinone and the respective dicopper(I) complex. The catalytic reaction follows a Michaelis–Menten behavior. The dicopper(I) complex binds molecular dioxygen at low temperature, forming a trans-μ-1,2-peroxo-dicopper adduct, which was characterized by UV–Vis and resonance Raman spectroscopy and electrochemically. This peroxo complex stoichiometrically oxidizes a second molecule of catechol in the absence of dioxygen. A catalytic mechanism of catechol oxidation by 1 has been proposed, and its relevance to the mechanisms earlier proposed for the natural enzyme and other copper complexes is discussed. [ABSTRACT FROM AUTHOR]

Hybrid density functional theory with the B3LYP functional has been used to investigate the catalytic mechanism of catechol oxidase. Catechol oxidase belongs to a class of enzymes that has a copper dimer with histidine ligands at the active site. Another member of this class is tyrosinase, which has been studied by similar methods previously. An important advantage for the present study compared to the one for tyrosinase is that X-ray crystal structures exist for catechol oxidase. The most critical step in the mechanism for catechol oxidase is where the peroxide O–O bond is cleaved. In the suggested mechanism this cleavage occurs in concert with a proton transfer from the substrate. Shortly after the transition state is passed there is another proton transfer from the substrate, which completes the formation of a water molecule. An important feature of the mechanism, like the one for tyrosinase, is that no proton transfers to or from residues outside the metal complex are needed. The calculated energetics is in reasonable agreement with experiments. Comparisons are made to other similar enzymes studied previously. [ABSTRACT FROM AUTHOR]

The preferred state, the peroxide Cu2(II,II) or the bis-µ-oxo Cu2(III,III) states, for oxygen-bridged copper dimers with nitrogen donors is reinvestigated. Experiments have indicated that for the enzymatic complexes with histidine ligands the peroxide state should be favored, at least for hemocyanin, while for the synthetic complexes with strained ligands the bis-µ-oxo state should be intrinsically favored. The present B3LYP study essentially agrees with these results. The quite different results obtained in CASPT2 and some previous B3LYP studies for these systems are investigated and discussed. The conclusion, drawn in an earlier study, that the Cu2(III,III) state is an unlikely intermediate in the enzyme mechanisms of tyrosinase and catechol oxidase, still remains. [ABSTRACT FROM AUTHOR]

The inhibition of the catechol oxidase activity exhibited by three dinuclear copper(II) complexes, derived from different diaminotetrabenzimidazole ligands, by kojic acid [5-hydroxy-2-(hydroxymethyl)- γ-pyrone] has been studied. The catalytic mechanism of the catecholase reaction proceeds in two steps and for both of these inhibition by kojic acid is of competitive type. The inhibitor binds strongly to the dicopper(II) complex in the first step and to the dicopper-dioxygen adduct in the second step, preventing in both cases the binding of the catechol substrate. Binding studies of kojic acid to the dinuclear copper(II) complexes and a series of mononuclear analogs, carried out spectrophotometrically and by NMR, enable us to propose that the inhibitor acts as a bridging ligand between the metal centers in the dicopper(II) catalysts. [ABSTRACT FROM AUTHOR]

Results from the identification of a collection of 43 table grape (Vitis vinifera L.) cultivars by isoenzymes from woody stems are presented. Plant extracts were separated by polyacrylamide gel electrophoresis. Isoenzyme analysis was carried out for peroxidases, catechol oxidase, glutamate oxalacetate transaminase, superoxide dismutase, esterases, and acid phosphatase. The last two systems were not adequate, while the rest showed a more or less marked polymorphism. Catechol oxidase was the system with the highest level of polymorphism. The combination of the zymograms allowed the separation of 31 out of the 43 studied cultivars. The other 12 cultivars were separated in five groups. The use of this methodology for differentiation of grapevine cultivars is recommended. [ABSTRACT FROM AUTHOR]

The cross-linking of protein molecules to form stable supramolecular aggregates capable of acting as protective and supporting structures is a common feature of organisms coping with the stresses of life. These new polymeric forms range from thick rigid structures to thin flexible membranes. The formation of such cross-links must be carefully controlled since more or less than optimal cross-linking could lead to malfunction or even death of the organism. The chemistry of the amino acids converted or directly involved in the formation of these cross-links is complex and a range of new amino acids has been identified. Di- and tri-tyrosines are formed by the action of peroxidases, quinones by catechol oxidases, γ glutamyl lysine iso-peptide bonds by glutamyl transferase and a complex series of lysine- aldehyde derived cross-links induced by lysyl oxidase. These cross-linking mechanisms provide an insight into the complex changes in tissue function during growth of the organism and their effects on the properties of foods. [ABSTRACT FROM AUTHOR]