EVIDENCE OF THE INDIRECT FORMATION OF THE CATECHOLIC INTERMEDIATE SUBSTRATE RESPONSIBLE FOR THE AUTOACTIVATION KINETICS OF TYROSINASE

Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the o xidation of monohydric phenol substrates consisting of a lag period th at increases with increasing substrate concentration, The cause of thi s is an autocatalytic process dependent on the generation of a dihydri c phenol substrate, which acts as an activator of the enzyme, Experime nts with N-substituted dihydric phenol substrate (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 peri od, N,N-Dipropyldopamine was oxidized with. normal kinetics but with a n 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 pro duct with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-cataly zed oxidation, Evidence that the indoliumolate product is formed by cy clization of the ortho-quinone is presented by pulse radiolysis studie s, which demonstrate the formation of the ortho-quinone (by disproport ionation of the corresponding semiquinones), which cyclizes to give th e indoliumolate, The rate constant for cyclization was shown to be 48 s(-1) (at pH 6.0), Tyrosinase-catalyzed oxidation of the monohydric ph enol analogue, N,N-dimethyltyramine, was shown to require the addition of a dihydric phenol, Oxygen utilization then exhibited a stoichiomet ry of 1.0, indicating that the reactions proceed only as far as the cy clization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to t he 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 indefi nite lag when N,N-dimethyltyramine is used as the substrate for tyrosi nase in the absence of a dihydric phenol cofactor.