Details

Autor(en) / Beteiligte

• Kuijpers, Tomas F. M.
• Gruppen, Harry
• Sforza, Stefano
• Berkel, Willem J. H.
• Vincken, Jean‐Paul

Titel

The antibrowning agent sulfite inactivates Agaricus bisporus tyrosinase through covalent modification of the copper‐B site

Ist Teil von

• The FEBS journal, 2013-12, Vol.280 (23), p.6184-6195

Ort / Verlag

England: Blackwell Publishing Ltd

Erscheinungsjahr

2013

Link zum Volltext

Quelle

Wiley Online Library

Beschreibungen/Notizen

• Sulfite salts are widely used as antibrowning agents in food processing. Nevertheless, the exact mechanism by which sulfite prevents enzymatic browning has remained unknown. Here, we show that sodium hydrogen sulfite (NaHSO3) irreversibly blocks the active site of tyrosinase from the edible mushroom Agaricus bisporus, and that the competitive inhibitors tropolone and kojic acid protect the enzyme from NaHSO3 inactivation. LC‐MS analysis of pepsin digests of NaHSO3‐treated tyrosinase revealed two peptides showing a neutral loss corresponding to the mass of SO3 upon MS2 fragmentation. These peptides were found to be homologous peptides containing two of the three histidine residues that form the copper‐B‐binding site of mushroom tyrosinase isoform PPO3 and mushroom tyrosinase isoform PPO4, which were both present in the tyrosinase preparation used. Peptides showing this neutral loss behavior were not found in the untreated control. Comparison of the effects of NaHSO3 on apo‐tyrosinase and holo‐tyrosinase indicated that inactivation is facilitated by the active site copper ions. These data provide compelling evidence that inactivation of mushroom tyrosinase by NaHSO3 occurs through covalent modification of a single amino‐acid residue, probably via addition of HSO3− to one of the copper‐coordinating histidines in the copper‐B site of the enzyme. Sulfite was found to irreversibly inactivate mushroom (Agaricus bisporus) tyrosinase. The competitive inhibitors tropolone and kojic acid protect the enzyme from inactivation, indicating that sulfite acts in the active site of tyrosinase. LC‐MS analysis of protease digests of sulfite–treated tyrosinase revealed that inactivation occurs through covalent modification of a single active site amino acid residue, most likely a copper‐coordinating histidine residue.