Abstract
Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).
Highlights
From the Department of Chemical and Biochemical Engineering and Center for Biocatalysis and Bioprocessing, Uniuersity of Iowa, Iowa City, Iowa 52242
One of the most significant limitations is the inability of Horseradish peroxidase (HRP) tocatalyze the modification of lignins that contain few free phenolic groups
The H202 generated by the nonenzymatic reactions catalyzes the formation of peroxidase compound I and leads directly to the peroxidatic oxidation of GSH to GS. as succinctly shown by the ability of HRP and Hz02 toform a DMPO spin adduct from the resulting thiyl radicals (Fig. 6)
Summary
Materials-Horseradish peroxidase (type II), bovine liver catalase, mM solution of veratryl alcohol was oxidized quantitatively superoxide dismutase from bovine blood, reduced glutathione, dithi- to veratraldehyde, and after 3 h, nearly 75% of the veratryl othreitol, dihydroxyfumaric acida,scorbic acid, L-cysteine,@-mercap- alcohol was oxidized quantitatively. Quantification of veratryl alcohol oxidation was followed by reverse K,,, for veratrylalcohol of 4.9 mM,apparent V,, of 0.18 pmol/. Veratryl alcohol oxidation was typically carried out i0n.1. With 1 mM veratryl alcohol, the apparent K, for GSH is5.2 mM. Time course HPLC studieswere carried out in 25-ml volumes and shaken on a rotary shaker a t 50 rpm a t 25 "C.Initial rate analyseswere performed in3-ml spectrophotometer cuvettes a t 25 "C. must react with HRP toform an activated complex that will react withasecond substrate [20]. The oxidation of the @-ether compounwdas followed by CI8reverse intersection of the plotof l / u uersu l/[veratryl alcohol] (Fig. phase HPLC with an eluentof 35% CH,CN in water anda flow rate 4) on they axis all indicate that HRP first reacwtsith GSH.
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