title: The pro-enzyme C-terminal processing domain of Pholiota nameko tyrosinase is responsible for folding of the N-terminal catalytic domain.
abstract: Pholiota nameko (Pholiota microspore) tyrosinase is expressed as a latent 67-kDa pro-tyrosinase, comprising a 42-kDa N-terminal catalytic domain with a binuclear copper centre and a 25-kDa C-terminal domain and is activated by proteolytic digestion of the C-terminal domain. To investigate the role of the C-terminal processing domain of pro-tyrosinase, we constructed a recombinant tyrosinase lacking the C-terminal domain and four recombinant pro-tyrosinase mutants (F515G, H539N, L540G and Y543G) carrying substituted amino acid residues on the C-terminal domain. The recombinant tyrosinase lacking the C-terminal domain had no catalytic activity; whereas the mutant L540G was copper depleted, the other mutants had copper contents similar to that of the wild-type pro-tyrosinase. Proteolytic digestion activated the mutants H539N and Y543G following release of the C-terminal domain, and the resulting tyrosinases had higher K m values for t-butyl catechol than the wild-type pro-tyrosinase. The mutants F515G and L540G were degraded by proteolytic digestion and yielded smaller proteins with no activity. These data suggest that the C-terminal processing domain of P. nameko pro-tyrosinase is essential for correct folding of the N-terminal catalytic domain and acts as an intramolecular chaperone during assembly of the active-site conformation.
authors.affiliation: Department of Food Science, Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa, 921-8836, Japan.
authors.forename: Lai Lai
authors.initials: LL
authors.lastname: Moe
authors.forename: Saya
authors.initials: S
authors.lastname: Maekawa
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Kawamura-Konishi
mesh_terms: D020134:Catalytic Domain; D002396:Catechols / Q000378:metabolism; D004252:DNA Mutational Analysis; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D055368:Pholiota / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002396:Catechols; D004792:Enzyme Precursors; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase; C034221:catechol