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The antibrowning agent sulfite inactivates Agaricus bisporus tyrosinase through covalent modification of the copper‐B site
Author(s) -
Kuijpers Tomas F. M.,
Gruppen Harry,
Sforza Stefano,
Berkel Willem J. H.,
Vincken JeanPaul
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12539
Subject(s) - tyrosinase , agaricus bisporus , chemistry , active site , kojic acid , sulfite , biochemistry , residue (chemistry) , enzyme , mushroom , food science
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 (Na HSO 3 ) 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 Na HSO 3 inactivation. LC ‐ MS analysis of pepsin digests of Na HSO 3 ‐treated tyrosinase revealed two peptides showing a neutral loss corresponding to the mass of SO 3 upon MS 2 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 Na HSO 3 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 Na HSO 3 occurs through covalent modification of a single amino‐acid residue, probably via addition of HSO 3 − to one of the copper‐coordinating histidines in the copper‐B site of the enzyme.