RESUMEN
Versatile peroxidase (VP) from Bjerkandera adusta is an enzyme able to oxidize bulky and high-redox substrates trough a Long-Range Electron Transfer (LRET) pathway. In this study, the introduction of radical-forming aromatic amino acids by chemical modification of the protein surface was performed, and the catalytic implications of these additional surface active-sites on the oxidation of 2,6-dimethylphenol, Mn2+ and Remazol Brilliant Blue R (RBBR) were determined. These three different substrates are oxidized in different active-sites of enzyme molecule, of which the high redox RBBR the only one that is transformed by an external radical formed on the protein surface. Both catalytic constants kcat and KM were significantly affected by the chemical modifications. Tryptophan- and tyrosine-modified VP showed higher catalytic transformation than the unmodified enzyme for RBBR, while the Mn2+ oxidation was significantly reduced by all chemical modifications. Electron Paramagnetic Resonance studies demonstrated the formation of additional protein-based radicals after the chemical modification with radical-forming amino acids. In addition, the catalytic rate of the LRET-mediated transformation showed a good correlation with the ionization energy of the additional amino acid on the protein surface.
Asunto(s)
Dominio Catalítico/genética , Coriolaceae/enzimología , Peroxidasa/genética , Peroxidasa/metabolismo , Antraquinonas/metabolismo , Transporte de Electrón , Cinética , Manganeso/metabolismo , Oxidación-Reducción , Xilenos/metabolismoRESUMEN
A Structure Activity Relationship (SAR) study for laccase mediator systems was performed in order to correctly classify different natural phenolic mediators. Decision tree (DT) classification models with a set of five quantum-chemical calculated molecular descriptors were used. These descriptors included redox potential (É°), ionization energy (E(i)), pK(a), enthalpy of formation of radical (Δ(f)H), and OH bond dissociation energy (D(O-H)). The rationale for selecting these descriptors is derived from the laccase-mediator mechanism. To validate the DT predictions, the kinetic constants of different compounds as laccase substrates, their ability for pesticide transformation as laccase-mediators, and radical stability were experimentally determined using Coriolopsis gallica laccase and the pesticide dichlorophen. The prediction capability of the DT model based on three proposed descriptors showed a complete agreement with the obtained experimental results.
Asunto(s)
Biocatálisis/efectos de los fármacos , Lacasa/metabolismo , Acetofenonas/química , Acetofenonas/farmacología , Benzaldehídos/química , Benzaldehídos/farmacología , Catecoles/química , Catecoles/farmacología , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacología , Árboles de Decisión , Diclorofeno/química , Diclorofeno/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hidrazonas/química , Hidrazonas/farmacología , Lacasa/química , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Nitrofenoles/química , Nitrofenoles/farmacología , Oxidación-Reducción , Fenoles/química , Fenoles/farmacología , Polyporales/enzimología , Conformación Proteica , Relación Estructura-Actividad Cuantitativa , Ácido Vanílico/química , Ácido Vanílico/farmacologíaRESUMEN
Three copper(II) complexes containing beta-cyclodextrin (betaCD) derivatives as ligands [mono-6-methylamino-6-deoxy-betaCD dithiocarbamate (CDTC), mono-6-histamino-6-deoxy-betaCD (CDHM) and mono-6-Nalpha-arginyl-6-deoxy-betaCD (CDARG)] have been studied by electron paramagnetic resonance. The spectra have been recorded at X- and S-bands and different temperatures and simulated to obtain the best set of magnetic parameters. In particular, the 300 K spectra are typical of the slow motion regime, as expected considering the high molecular weight of the ligands. Some structural characteristics of the complexes are proposed on the basis of dynamic and covalency parameters obtained from simulation.