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 µM. 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 µM. 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.
Skip Nav Destination
Article navigation
August 1998
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
Research Article|
August 01 1998
Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate
Christopher J. COOKSEY;
Christopher J. COOKSEY
*Department of Chemistry, Christopher Ingold Laboratories, UCL, 20 Gordon Street, London WC1H 0AJ, U.K.
Search for other works by this author on:
Peter J. GARRATT;
Peter J. GARRATT
*Department of Chemistry, Christopher Ingold Laboratories, UCL, 20 Gordon Street, London WC1H 0AJ, U.K.
Search for other works by this author on:
Edward J. LAND;
Edward J. LAND
†CRC Section of Drug Development and Imaging, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 9BX, U.K.
Search for other works by this author on:
Christopher A. RAMSDEN;
Christopher A. RAMSDEN
‡Department of Chemistry, Keele University, Keele, Staffs. ST5 5BG, U.K.
Search for other works by this author on:
Patrick A. RILEY
Patrick A. RILEY
1
§Department of Molecular Pathology, Windeyer Institute for Medical Research, UCL Medical School, Cleveland Street, London W1P 6DB, U.K.
1To whom correspondence should be addressed (e-mail: p.riley@ucl.ac.uk).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
February 19 1998
Revision Received:
April 21 1998
Accepted:
May 11 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 333 (3): 685–691.
Article history
Received:
February 19 1998
Revision Received:
April 21 1998
Accepted:
May 11 1998
Citation
Christopher J. COOKSEY, Peter J. GARRATT, Edward J. LAND, Christopher A. RAMSDEN, Patrick A. RILEY; Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate. Biochem J 1 August 1998; 333 (3): 685–691. doi: https://doi.org/10.1042/bj3330685
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.