Abstract
We have investigated the lignin peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. Pre-steady-state kinetic analyses demonstrated that guaiacol is a good substrate for both compounds I and II, the two- and one-electron oxidized enzyme intermediates, respectively, of lignin peroxidase. The rate constant for the reaction with compound I is 1.2 x 10(6) M-1s-1. The reaction of guaiacol with compound II exhibits a Kd of 64 microM and a first-order rate constant of 17 s-1. Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with a Km of 160 microM and a kcat of 7.7 s-1. Veratryl alcohol, a secondary metabolite of ligninolytic fungi, is capable of mediating the oxidation of guaiacol. This was shown by steady-state inhibition studies. Guaiacol completely inhibited the oxidation of veratryl alcohol, whereas veratryl alcohol had no corresponding inhibitory effect on guaiacol oxidation. In fact, at low guaiacol concentrations, veratryl alcohol stimulated the rate of guaiacol oxidation. These results collectively demonstrate that veratryl alcohol can serve as a mediator for phenolic substrates in the lignin peroxidase reaction.
Highlights
We have investigated the lignin peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods
These workers observed that substrates that are not oxidized by lignin peroxidase such as anisyl alcohol and 4-methoxymandelic acid are oxidized in the presence of veratryl alcohol [12]
Steady-state Kinetics of Guaiacol Oxidation—High pressure liquid chromatography analysis demonstrated that the oxidation of guaiacol by lignin peroxidase produces tetraguaiacol, as it does for horseradish peroxidase [22]
Summary
We have investigated the lignin peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. These workers observed that substrates that are not oxidized by lignin peroxidase such as anisyl alcohol and 4-methoxymandelic acid are oxidized in the presence of veratryl alcohol [12] They proposed that the one-electron oxidized product of veratryl alcohol, the aryl cation radical, is able to mediate the oxidation of substrates typically not oxidized by the enzyme. In contrast to Harvey et al [12], Valli et al [13] proposed that the stimulation of 4-methoxymandelic acid and anisyl alcohol oxidation is due solely to the ability of veratryl alcohol to prevent inactivation of lignin peroxidase They claimed that enzyme in the presence of anisyl alcohol and excess H2O2 leads to the formation of inactive compound III* [13]. We show that guaiacol, like veratryl alcohol, is capable of converting the enzyme from the compound III state back to the resting ferric state in the presence of H2O2
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