Oxidation of 4-tert-butylcatechol and dopamine by hydrogen peroxide catalysed by horseradish peroxidase.
The catalytic cycle of horseradish peroxidase (HRP; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) is initiated by a rapid oxidation of it by hydrogen peroxide to give an enzyme intermediate, compound I, which reverts to the resting state via two successive single electron transfer reactions from reducing substrate molecules, the first yielding a second enzyme intermediate, compound II. To investigate the mechanism of action of horseradish peroxidase on catechol substrates we have studied the oxidation of both 4-tert-butylcatechol and dopamine catalysed by this enzyme. The different polarity of the side chains of both o-diphenol substrates could help in the understanding of the nature of the rate-limiting step in the oxidation of these substrates by the enzyme. The procedure used is based on the experimental data to the corresponding steady-state equations and permitted evaluation of the more significant individual rate constants involved in the corresponding reaction mechanism. The values obtained for the rate constants for each of the two substrates allow us to conclude that the reaction of horseradish peroxidase compound II with o-diphenols can be visualised as a two-step mechanism in which the first step corresponds to the formation of an enzyme-substrate complex, and the second to the electron transfer from the substrate to the iron atom. The size and hydrophobicity of the substrates control their access to the hydrophobic binding site of horseradish peroxidase, but electron density in the hydroxyl group of C-4 is the most important feature for the electron transfer step.
García-Moreno
M
M
Departamento de Química-Fisica, Escuela Politécnica Superior, Universidad de Castilla-La Mancha, Albacete, Spain.
Moreno-Conesa
M
M
Rodríguez-López
J N
JN
García-Cánovas
F
F
Varón
R
R
D002384:Catalysis; D002396:Catechols / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004298:Dopamine / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D002396:Catechols; C038096:tert-butylcatechol; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D004298:Dopamine