title: Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase.
abstract: Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the oxidation of monohydric phenol substrates consisting of a lag period that increases with increasing substrate concentration. The cause of this is an autocatalytic process dependent on the generation of a dihydric phenol substrate, which acts as an activator of the enzyme. Experiments with N-substituted dihydric phenol substrates (N-methyldopamine, N-acetyldopamine) demonstrate that oxygen consumption is retarded in the N-acetyl substituted material due to a diminished rate of cyclization. The oxygen uptake exhibited a similar pattern when N-acetyltyramine was oxidized, and this was reflected by a prolongation of the lag period. N,N-Dipropyldopamine was oxidized with normal kinetics but with an oxygen stoichiometry of 0.5 mol of oxygen/mol of substrate. We show that this is the result of the formation of a stable indoliumolate product with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-catalyzed oxidation. Evidence that the indoliumolate product is formed by cyclization of the ortho-quinone is presented by pulse radiolysis studies, which demonstrate the formation of the ortho-quinone (by disproportionation of the corresponding semiquinones), which cyclizes to give the indoliumolate. The rate constant for cyclization was shown to be 48 s-1 (at pH 6.0). Tyrosinase-catalyzed oxidation of the monohydric phenol analogue, N, N-dimethyltyramine, was shown to require the addition of a dihydric phenol. Oxygen utilization then exhibited a stoichiometry of 1.0, indicating that the reactions proceed only as far as the cyclization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to the indoliumolate derived from N,N-dipropyldopamine. This material was methylated by catechol O-methyltransferase but was unreactive to redox reagents. The formation of the cyclic product accounts for the indefinite lag when N,N-dimethyltyramine is used as the substrate for tyrosinase in the absence of a dihydric phenol cofactor.
authors.affiliation: Department of Chemistry, Christopher Ingold Laboratories, UCL, 20 Gordon Street, London WC1H 0AJ, United Kingdom.
authors.forename: C J
authors.initials: CJ
authors.lastname: Cooksey
authors.forename: P J
authors.initials: PJ
authors.lastname: Garratt
authors.forename: E J
authors.initials: EJ
authors.lastname: Land
authors.forename: S
authors.initials: S
authors.lastname: Pavel
authors.forename: C A
authors.initials: CA
authors.lastname: Ramsden
authors.forename: P A
authors.initials: PA
authors.lastname: Riley
authors.forename: N P
authors.initials: NP
authors.lastname: Smit
mesh_terms: D002396:Catechols / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D019800:Phenol / Q000737:chemistry / Q000378:metabolism; D011675:Pulse Radiolysis; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D002396:Catechols; D019800:Phenol; D014442:Monophenol Monooxygenase