Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones

• Published in: Biochimica et biophysica acta Vol. 1548; no. 1; pp. 1 - 22
• Main Authors: Fenoll, Lorena G., Rodrı́guez-López, José Neptuno, Garcı́a-Sevilla, Francisco, Garcı́a-Ruiz, Pedro Antonio, Varón, Ramón, Garcı́a-Cánovas, Francisco, Tudela, José
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 09.07.2001
• Subjects: Agaricales - enzymology; Catechol Oxidase - chemistry; Dose-Response Relationship, Drug; Enzyme kinetics; Kinetics; Levodopa - chemistry; Models, Chemical; Models, Theoretical; Monophenol; Monophenol Monooxygenase - chemistry; Monophenol Monooxygenase - pharmacology; Mushroom; Nuclear magnetic resonance; Numerical integration; o-Diphenol; o-Quinone; Phenols - chemistry; Polyphenol oxidase; Quinones - chemistry; Reaction mechanism; Spectrophotometry; Tyrosinase; Tyrosine - chemistry; E m, Mettyrosinase (with Cu 2+-Cu 2+ in the active site); o-Quinone; Cr, aminochrome; Tyrosinase or polyphenol oxidase, monophenol, ortho-diphenol: oxygen oxidoreductase, EC 1.14.18.1; δ 3, chemical displacement value at C-3; E ox, Oxytyrosinase with Cu 2+- O 2 2−-Cu 2+ in the active site; L, leucoaminochrome; K 2= k −2/ k +2; Q, o-quinone; [D 0] ss, o-diphenol concentration in the first pseudo-steady state when tyrosinase acts on M; C-3, carbon atom in the 3-position of the benzene ring; [D f] ss, o-diphenol concentration in the second steady state when tyrosinase acts on M; V ss1 M, initial pseudo-steady-state rate of [QH]+[Cr] accumulation when tyrosinase acts on M; Reaction mechanism; Nuclear magnetic resonance; K 1= k −1/ k +1; Monophenol; [M] 0, initial monophenol concentration; Numerical integration; NMR, Nuclear Magnetic Resonance; o-Diphenol; λ i, wavelength at the isosbestic point between QH and Cr; Mushroom; QH, o-quinone protonated; D, o-diphenol; C-4, carbon atom in the 4-position of the benzene ring; δ 4, chemical displacement value at C-4; [E] 0 initial tyrosinase concentration; ε i, molar absorptivity at λ i; M, monophenol; V ss2 M, final steady-state rate of [QH]+[Cr] accumulation when tyrosinase acts on M; [D] 0, initial o-diphenol concentration; V ss D, steady-state rate of [QH]+[Cr] accumulation when tyrosinase acts on D; Enzyme kinetics; Tyrosinase; Polyphenol oxidase; E d, Deoxytyrosinase (with Cu 1+-Cu 1+ in the active site)
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(01)00207-2

Tyrosinase can act on monophenols because of the mixture of met- (E m) and oxy-tyrosinase (E ox) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as l-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D 0] ss. Hence, the system reaches an initial pseudo-steady state when t→0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D f] ss. These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, E mD, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E ox on monophenols, E mD, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D f] ss) necessary for the final steady state to be reached after the lag period.