Original Research Article Rate Constants for the First Two Chemical Steps of Eumelanogenesis E. J. LAND 1 , S. ITO 2 , K. WAKAMATSU 2 and P. A. RILEY 3 1 Department of Chemistry and Physics, Keele University, Keele, UK; 2 Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan; 3 The Totteridge Institute for Advanced Studies, 2 The Grange, Grange Avenue, London, UK *Address reprint requests to P.A. Riley, Totteridge Institute, 2 The Grange, Grange Avenue, London N20 8AB, UK. E-mail: rebc900@ucl.ac.uk Received 30 April 2003; in final form 19 June 2003 The kinetics of the initial cyclization and redox exchange reactions involved in the eumelanogenic pathway have been studied previously but because of the difficulty of detecting the intermediate cyclodopa by optical means (because its absorb- ance is in the same range as dopa which is present in excess in the experimental system) no accurate value for the redox exchange reaction has so far been obtained and there is no available analytical methodology that can be applied to the successive first- and second-order reactions involved. We have synthesized cyclodopa and examined the kinetics of the formation of dopachrome following the pulse radiolytic generation of dopaquinone in its presence. From this direct measurement we determined that the rate constant of the reaction between cyclodopa and dopaquinone is 5.3 · 10 6 /M/s. Employing this value in a computational model of the combined cyclization and redox exchange reactions we calculate that the observed kinetics of dopaquinone decay and dopachrome formation are compatible with a cyclization rate constant of 3.8/s. Key words: Cyclodopa, Redox exchange, Pulse radiolysis, Computer model, Dopaquinone, Dopachrome INTRODUCTION The initial reactions in the biosynthesis of eumelanin involve a tyrosinase-catalysed oxidation of tyrosine or 3,4-dihydroxyphenylalanine (dopa) to yield dopaquinone followed by two apparently spontaneous reactions: the cyclization of the quinone to give cyclodopa and the subsequent redox exchange between dopaquinone and cyclodopa to give rise to dopachrome and regenerating dopa. The cyclization reaction is a first-order reaction in which there is an intramolecular Michael addition reaction in which the lone pair electrons of the nitrogen atom in the side-chain amino group complete a heterocyclic secondary ring. This nucleophilic addition reaction is inhibited by increasing the proton concentration but takes place with a reported rate constant of 7.6/s (1). The subsequent redox exchange reaction is rapid but has not hitherto been measured directly. In the paper by Chedekel et al. (1) the rate constant of the redox exchange reaction was assumed to be ‡10 9 /M/s but subsequent work (2) suggested that the redox exchange reaction was slower than previously thought, with an upper limit of 4 · 10 7 /M/s. Because it is a second-order process dependent on the initial first-order reaction to supply the reducing substrate, there is no simple method of estimating the rate constant as the corresponding differential equations do not have an explicit solution (3). We, therefore, synthesized cyclodopa and this paper reports the results of the direct measurement of the rate constant for the redox exchange reaction and the subsequent estimation of the cyclization rate constant from a compu- tational model of the combined cyclization and redox exchange reactions and the apparent half lives of dopaqui- none decay and dopachrome formation. MATERIALS AND METHODS Chemicals Cyclodopa was synthesized by the method of Wyler and Chiovini (4). A good yield of triacetylcyclodopa methyl ester was obtained and crystallized. This material was heated in 6 M HCl under vacuum at 80° for 20 h. and the PIGMENT CELL RES 16: 487–493. 2003 Copyright Ó Blackwell Munksgaard 2003 Printed in UK—all rights reserved ISSN 0893-5785 Pigment Cell Res. 16, 2003 487