Agaricus bisporus metapotyrosinase: preparation, characterization, and conversion to mixed-metal derivatives of the binuclear site

Abstract

The alpha, beta, and gamma isozymes of Agaricus bisporus tyrosinase undergo inactivation in the presence of oxalate. The inactivation rate law is first order in enzyme and second order in oxalate. On a more rapid time scale than inactivation, oxalate acts as a competitive inhibitor of the catecholase reaction of tyrosinase. After removal of oxalate by dialysis, the inactivated enzyme is found to contain 50% of the original copper, all of which is present as paramagnetic, mononuclear copper sites. The ESR parameters of this copper indicate a tetragonal environment with nitrogen and oxygen ligands. The product of oxalate inactivation has lost one copper from each binuclear site and is thus a metapo derivative. Addition of Cu(II) to metapotyrosinase results in complete recovery of copper and catalytic activity. Prolonged storage of metapotyrosinase, in the absence of any additional Cu(II), results in copper migration, producing a 50% recovery of the original specific activity, expressed on a protein basis. Copper migration converts metapo sites into equal numbers of reconstituted, holo sites and fully apo sites. Both copper migration and copper reconstitution follow apparent first-order kinetics and are pH dependent. The involvement of two ionizable groups accounts for the observed pH dependence of each process. For copper migration pKa values of 6.0 and 8.8 were found, while for copper reconstitution the pKa values were 5.4 and 6.9. Addition of either Co(II) or Zn(II) to metapotyrosinase results in the formation of enzymatically inactive, mixed-metal derivatives of the binuclear copper site having one Cu(II) and one Co(II) or Zn(II) ion.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Sodium Periodate and Tyrosinase on Binding of Alginate to Adlayers of Mytilus edulis Foot Protein 1

“…Mushroom tyrosinase is expected to bind to the catechol functionality of L-dopa residues by coordination of the oxygens to a binuclear copper site (30,31). Adlayers of Mefp-1 promote binding of mushroom tyrosinase (compared to a clean Ge substratum).…”

Comparative evaluation of rosmarinic acid, methyl rosmarinate and pedalitin isolated from Rabdosia serra (MAXIM.) HARA as inhibitors of tyrosinase and α-glucosidase

“…Tyrosinase is responsible for enzymatic browning in plants. Tyrosinase, is a copper-containing enzyme that catalyses the hydroxylation of a monophenol and the oxidation of o-diphenols to o-quinones (Yong, Leone, & Strothkamp, 1990). Additionally, quinones reacted with amines, amino acids, peptides and proteins could result in a loss of nutritional quality, decrease of digestibility and inhibition of proteolytic and glycolytic enzymes (Kim & Uyama, 2005).…”

Kinetics study of the oxidation of 4‐tert‐butylphenol by tyrosinase

“…Mushroom tyrosinase has a molecular mass of 120 kDa. It is composed of two H subunits (43 kDa) and two L subunits (13 kDa) and contains two binuclear active sites/molecule [6]. Most of the enzyme in a fresh preparation is in the met-tyrosinase form, in which the active site is bicupric and unable to bind oxygen.…”