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 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).
Division of Advanced Science and Biotechnology, Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan. fujieda@mls.eng.osaka-u.ac.jp
Nobutaka
N
Fujieda
Michiaki
M
Murata
Shintaro
S
Yabuta
Takuya
T
Ikeda
Chizu
C
Shimokawa
Yukihiro
Y
Nakamura
Yoji
Y
Hata
Shinobu
S
Itoh
D001236:Aspergillus oryzae / Q000201:enzymology*; D020134:Catalytic Domain; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D008958:Models, Molecular; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism
D010636:Phenols; D014442:Monophenol Monooxygenase