Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate.

2.50
Hdl Handle:
http://hdl.handle.net/10541/93000
Title:
Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate.
Authors:
Cooksey, C J; Garratt, P J; Land, Edward J; Ramsden, C A; Riley, P A
Abstract:
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.
Affiliation:
Department of Chemistry, Christopher Ingold Laboratories, UCL, 20 Gordon Street, London WC1H 0AJ, UK.
Citation:
Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate. 1998, 333 ( Pt 3):685-91 Biochem. J.
Journal:
The Biochemical Journal
Issue Date:
1-Aug-1998
URI:
http://hdl.handle.net/10541/93000
PubMed ID:
9677329
Type:
Article
Language:
en
ISSN:
0264-6021
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorCooksey, C Jen
dc.contributor.authorGarratt, P Jen
dc.contributor.authorLand, Edward Jen
dc.contributor.authorRamsden, C Aen
dc.contributor.authorRiley, P Aen
dc.date.accessioned2010-02-25T11:20:34Z-
dc.date.available2010-02-25T11:20:34Z-
dc.date.issued1998-08-01-
dc.identifier.citationTyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate. 1998, 333 ( Pt 3):685-91 Biochem. J.en
dc.identifier.issn0264-6021-
dc.identifier.pmid9677329-
dc.identifier.urihttp://hdl.handle.net/10541/93000-
dc.description.abstractWhen 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.en
dc.language.isoenen
dc.subject.meshAcetonitriles-
dc.subject.meshCatechols-
dc.subject.meshEnzyme Activation-
dc.subject.meshHumans-
dc.subject.meshHydrogen-Ion Concentration-
dc.subject.meshKinetics-
dc.subject.meshMiddle Aged-
dc.subject.meshMonophenol Monooxygenase-
dc.subject.meshNitriles-
dc.subject.meshOxidation-Reduction-
dc.subject.meshQuinones-
dc.subject.meshSpectrophotometry-
dc.titleTyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate.en
dc.typeArticleen
dc.contributor.departmentDepartment of Chemistry, Christopher Ingold Laboratories, UCL, 20 Gordon Street, London WC1H 0AJ, UK.en
dc.identifier.journalThe Biochemical Journalen
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