Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones Lorena G. Fenoll a , Jose ¨ Neptuno Rodr| ¨guez-Lo ¨pez a , Francisco Garc| ¨a-Sevilla b , Pedro Antonio Garc| ¨a-Ruiz c , Ramo ¨n Varo ¨n b , Francisco Garc| ¨a-Ca ¨novas a ; *, Jose ¨ Tudela a a GENZ: Grupo de Investigacio ¨n Enzimolog| ¨a, Departamento de Bioqu| ¨mica y Biolog| ¨a Molecular-A, Facultad de Biolog| ¨a, A. Correos 4021 Universidad de Murcia, E-30100 Murcia, Spain b Departamento de Qu| ¨mica-F| ¨sica, Escuela Te ¨cnica Superior de Albacete, Universidad de Castilla-La Mancha, Castilla-La Manch, Spain c Departamento de Qu| ¨mica Orga ¨nica, Facultad de Qu| ¨mica, Universidad de Murcia, Murcia, Spain Received 19 October 2000; received in revised form 27 March 2001; accepted 12 April 2001 Abstract 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 tC0 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 m D, 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 m D, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the 0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII:S0167-4838(01)00207-2 Abbreviations: C-3, carbon atom in the 3-position of the benzene ring; C-4, carbon atom in the 4-position of the benzene ring; Cr, aminochrome ; N 3 , chemical displacement value at C-3; N 4 , chemical displacement value at C-4; D, o-diphenol; [D] 0 , initial o-diphenol concentration ; [D 0 ] ss , o-diphenol concentration in the ¢rst pseudo-steady state when tyrosinase acts on M; [D f ] ss , o-diphenol concen- tration in the second steady state when tyrosinase acts on M; L, leucoaminochrome; M, monophenol; [M] 0 , initial monophenol con- centration ; V i , wavelength at the isosbestic point between QH and Cr; A i , molar absorptivity at V i ; [E] 0 initial tyrosinase concentration ; E d , Deoxytyrosinase (with Cu 1 -Cu 1 in the active site); E m , Mettyrosinase (with Cu 2 -Cu 2 in the active site); E ox , Oxytyrosinase with Cu 2 -O 23 2 -Cu 2 in the active site; K 1 = k 31 /k 1 ; K 2 = k 32 /k 2 ; Q, o-quinone; QH, o-quinone protonated ; NMR, Nuclear Magnetic Resonance ; V D ss , steady-state rate of [QH]+[Cr] accumulation when tyrosinase acts on D; V M ss1 , initial pseudo-steady-state rate of [QH]+[Cr] accumulation when tyrosinase acts on M; V M ss2 , ¢nal steady-state rate of [QH]+[Cr] accumulation when tyrosinase acts on M ; Tyrosinase or polyphenol oxidase, monophenol, ortho-diphenol : oxygen oxidoreductase, EC 1.14.18.1 * Corresponding author. Fax: +34-968-364147; E-mail: canovasf@um.es Biochimica et Biophysica Acta 1548 (2001) 1^22