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.
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- Cooksey C. J., Garratt P. J., Land E. J., Pavel S., Ramsden C. A., Riley P. A., Smit N. P. Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase. J Biol Chem. 1997 Oct 17;272(42):26226–26235. doi: 10.1074/jbc.272.42.26226. [DOI] [PubMed] [Google Scholar]
- Evans W. C., Raper H. S. The accumulation of l-3:4-dihydroxyphenylalanine in the tyrosinase-tyrosine reaction. Biochem J. 1937 Dec;31(12):2162–2170. doi: 10.1042/bj0312162. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lerch K. Neurospora tyrosinase: structural, spectroscopic and catalytic properties. Mol Cell Biochem. 1983;52(2):125–138. doi: 10.1007/BF00224921. [DOI] [PubMed] [Google Scholar]
- Naish-Byfield S., Cooksey C. J., Riley P. A. Oxidation of monohydric phenol substrates by tyrosinase: effect of dithiothreitol on kinetics. Biochem J. 1994 Nov 15;304(Pt 1):155–162. doi: 10.1042/bj3040155. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Naish-Byfield S., Riley P. A. Oxidation of monohydric phenol substrates by tyrosinase. An oximetric study. Biochem J. 1992 Nov 15;288(Pt 1):63–67. doi: 10.1042/bj2880063. [DOI] [PMC free article] [PubMed] [Google Scholar]
- OSAKI S. The mechanism of tyrosine oxidation by mushroom tyrosinase. Arch Biochem Biophys. 1963 Mar;100:378–384. doi: 10.1016/0003-9861(63)90101-2. [DOI] [PubMed] [Google Scholar]
- Palumbo A., d'Ischia M., Misuraca G., Carratú L., Prota G. Activation of mammalian tyrosinase by ferrous ions. Biochim Biophys Acta. 1990 Mar 26;1033(3):256–260. doi: 10.1016/0304-4165(90)90129-k. [DOI] [PubMed] [Google Scholar]
- Solomon E. I., Lowery M. D. Electronic structure contributions to function in bioinorganic chemistry. Science. 1993 Mar 12;259(5101):1575–1581. doi: 10.1126/science.8384374. [DOI] [PubMed] [Google Scholar]
- Sugumaran M., Semensi V., Kalyanaraman B., Bruce J. M., Land E. J. Evidence for the formation of a quinone methide during the oxidation of the insect cuticular sclerotizing precursor 1,2-dehydro-N-acetyldopamine. J Biol Chem. 1992 May 25;267(15):10355–10361. [PubMed] [Google Scholar]
- Sánchez-Ferrer A., Rodríguez-López J. N., García-Cánovas F., García-Carmona F. Tyrosinase: a comprehensive review of its mechanism. Biochim Biophys Acta. 1995 Feb 22;1247(1):1–11. doi: 10.1016/0167-4838(94)00204-t. [DOI] [PubMed] [Google Scholar]