Acid activation of protyrosinase from Aspergillus oryzae: homo-tetrameric protyrosinase is converted to active dimers with an essential intersubunit disulfide bond at acidic pH.
Aspergillus oryzae protyrosinase (pro-TY) has a unique feature that the proenzyme is activated under conditions of acidic pH. The pro-TY was inactive at pH 7.0. The latent enzyme was activated at pH 3.0, and was slightly activated by sodium dodecyl sulfate (SDS). The molecular masses of the pro-TY and acid-activated tyrosinase (acid-TY) were 266 and 165 kDa, respectively, as estimated by gel-filtration chromatography. The CD spectra showed that the tertiary and/or quaternary structure was changed after the acid activation. On the basis of these results, we deduce that the intersubunit polar interaction is disrupted at pH 3.0, and that the tetrameric pro-TY dissociates to dimers. Tryptophan fluorescence spectra and binding assay of 8-anilino-1-naphthalene sulfonic acid (ANS) suggested that hydrophobic amino acid residues of the active site were exposed to solvent after acid treatment. It was likely that Cys108 formed an intermolecular disulfide bond between the subunits of dimeric acid-TY. The dimerization of acid-TY involving the intermolecular disulfide bond is essential for the activity.
Tatara
Yota
Y
Laboratory of Molecular Enzymology, Graduate School of Bioengineering, Soka University, Hachioji, Tokyo, Japan.
Namba
Takeshi
T
Yamagata
Youhei
Y
Yoshida
Takashi
T
Uchida
Takafumi
T
Ichishima
Eiji
E
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013050:Spectrometry, Fluorescence
C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; D005656:Fungal Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; C092659:protyrosinase protein, Aspergillus oryzae; D012967:Sodium Dodecyl Sulfate; D003545:Cysteine