Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate.
When 3,4-dihydroxybenzylcyanide (DBC) is oxidized by mushroom tyrosinase, the first visible product, identified as the corresponding quinomethane, exhibits an absorption maximum at 480 nm. Pulse-radiolysis experiments, in which the o-quinone is formed by disproportionation of semiquinone radicals generated by single-electron oxidation of DBC, showed that the quinomethane (A480 6440 M-1.cm-1) is formed through the intermediacy of the o-quinone with a rate constant at neutral pH of 7.5 s-1. The oxygen stoichiometry of the formation of the quinomethane by tyrosinase-catalysed oxidation of DBC was 0.5:1. On the basis of oxygen utilization rates the calculated Vmax was 4900 nmol.min-1 and the apparent Km was 374 microM. The corresponding monohydric phenol, 4-hydroxybenzylcyanide (HBC), was not oxidized by tyrosinase unless the enzyme was pre-exposed to DBC, the maximum acceleration of HBC oxidation being obtained by approximately equimolar addition of DBC. These results are consistent with tyrosinase auto-activation on the basis of the indirect formation of the dihydric phenol-activating cofactor. The rapid conversion of the o-quinone to the quinomethane prevents the formation of the catechol by reduction of the o-quinone product of monohydric phenol oxidation from occurring in the case of the compounds studied. In the absence of auto-activation, the kinetic parameters for HBC oxidation by tyrosinase were estimated as Vmax 70 nmol.min-1 and Km 309 microM. The quinomethane was found to decay with a rate constant of 2k 38 M-1.s-1, as determined both by pulse-radiolysis and tyrosinase experiments. The second-order kinetics indicate that a dimer is formed. In the presence of tyrosinase, but not in the pulse-radiolysis experiments, the quinomethane decay was accompanied by a steady-state oxygen uptake concurrently with the generation of a melanoid product measured by its A650, which is ascribed to the formation of an oligomer incorporating the oxidized dimer.
Department of Chemistry, Christopher Ingold Laboratories, UCL, 20 Gordon Street, London WC1H 0AJ, UK.
C J
CJ
Cooksey
P J
PJ
Garratt
E J
EJ
Land
C A
CA
Ramsden
P A
PA
Riley
D000097:Acetonitriles / Q000737:chemistry*; D002396:Catechols / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008875:Middle Aged; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D009570:Nitriles / Q000138:chemical synthesis* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011809:Quinones / Q000737:chemistry*; D013053:Spectrophotometry
C114328:3,4-dihydroxybenzylcyanide; D000097:Acetonitriles; D002396:Catechols; D009570:Nitriles; D011809:Quinones; C026914:4-hydroxybenzylcyanide; D014442:Monophenol Monooxygenase; C034221:catechol