Tyrosinase action on monophenols: evidence for direct enzymatic release of o-diphenol.
Using gas chromatography-mass spectrometry, the direct enzymatic release of o-diphenol (4-tert-butylcatechol) during the action of tyrosinase on a monophenol (4-tert-butylphenol) has been demonstrated for the first time in the literature. The findings confirm the previously proposed mechanism to explain the action of tyrosinase on monophenols (J.N. Rodríguez-López, J. Tudela, R. Varón, F. García-Carmona, F. García-Cánovas, J. Biol. Chem. 267 (1992)). Oxytyrosinase, the oxidized form of the enzyme with a peroxide group, is the only form capable of catalysing the transformation of monophenols into diphenols, giving rise to an enzyme-substrate complex in the process. The o-diphenol formed is then released from the enzyme-substrate complex or oxidized to the corresponding o-quinone. In order to detect the enzymatic release of o-diphenol, the non-enzymatic evolution of the o-quinone to generate o-diphenol by weak nucleophilic attack reactions and subsequent oxidation-reduction was blocked by the nucleophilic attack of an excess of cysteine. Furthermore, the addition of catalytic quantities of an auxiliary o-diphenol (e.g. catechol) considerably increases the accumulation of 4-tert-butylcatechol. The enzyme acting on 4-tert-butylphenol generates the enzyme-4-tert-butylcatechol complex and 4-tert-butylcatechol is then released (with k(-2)) generating mettyrosinase. The auxiliary o-diphenol added (catechol) and the 4-tert-butylcatechol generated by the enzyme then enter into competition. When [catechol] >> [4-tert-butylcatechol], the enzyme preferentially binds with the catechol to close the catalytic cycle, while 4-tert-butylcatechol is accumulated in the medium. In conclusion, we demonstrate that the enzyme produces 4-tert-butylcatechol from 4-tert-butylphenol, the concentration of which increases considerably in the presence of an auxiliary o-diphenol such as catechol.
Rodríguez-López
J N
JN
Grupo Investigación Enzimologia, Departmento de Bioquímica y Biologia Molecular-A, Facultad de Biologia, Univesidad de Murcia, Spain.
Fenoll
L G
LG
Peñalver
M J
MJ
García-Ruiz
P A
PA
Varón
R
R
Martínez-Ortíz
F
F
García-Cánovas
F
F
Tudela
J
J
D000363:Agaricales; D016227:Benzoquinones / Q000737:chemistry; D002396:Catechols / Q000737:chemistry* / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000494:pharmacology; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D007700:Kinetics; D020460:Melanosomes / Q000378:metabolism; D008956:Models, Chemical; D008962:Models, Theoretical; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010636:Phenols / Q000737:chemistry*
D016227:Benzoquinones; D002396:Catechols; D010636:Phenols; C015047:p-tert-butyl catechol; D014442:Monophenol Monooxygenase; D003545:Cysteine; C006862:butylphen