RESUMO
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 O(2)-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).
Assuntos
Aspergillus oryzae/enzimologia , Monofenol Mono-Oxigenase/metabolismo , Domínio Catalítico , Ativação Enzimática , Concentração de Íons de Hidrogênio , Hidroxilação , Modelos Moleculares , Monofenol Mono-Oxigenase/química , Fenóis/metabolismoRESUMO
The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH(2)OH (reductant), the proenzyme bound dioxygen to give a stable (µ-η(2):η(2) -peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458-Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.
Assuntos
Aspergillus oryzae/enzimologia , Monofenol Mono-Oxigenase/metabolismo , Sequência de Aminoácidos , Animais , Dimerização , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Moluscos/química , Moluscos/enzimologia , Moluscos/genética , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Autocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with Cu(II) under aerobic conditions, where a (µ-η(2):η(2)-peroxo)dicopper(II) species has been suggested to be involved as a key reactive intermediate.
