초록
<P><B>Highlights</B></P><P>► Glyoxal oxidase of the fungus <I>P. chrysosporium</I> can accept glycerol as substrate. ► Glyoxal oxidase inactivation during enzyme turnover prevents substrate conversion. ► Addition of horseradish peroxidase, catalase and a chemical oxidant partially avoids inactivation. ► The three-enzyme system allows the two-step conversion of glycerol to glyceric acid.</P> <P><B>Abstract</B></P><P>Glyoxal oxidase of <I>P. chrysosporium</I> is a radical copper oxidase that catalyzes oxidation of aldehydes to carboxylic acids coupled to dioxygen reduction to H<SUB>2</SUB>O<SUB>2</SUB>. In addition to known substrates, glycerol is also found to be a substrate for glyoxal oxidase. During enzyme turnover, glyoxal oxidase undergoes a reversible inactivation, probably caused by loss of the active site free radical, resulting in short-lasting enzyme activities and undetectable substrate conversions. Enzyme activity could be extended by including two additional enzymes, horseradish peroxidase and catalase, in addition to a redox chemical activator, such as Mn(III) (or Mn(II)+H<SUB>2</SUB>O<SUB>2</SUB>) or hexachloroiridate. Using this three-enzyme system glycerol was converted in glyceric acid in a two-step reaction, with glyceraldehyde as intermediate. A possible operation mechanism is proposed in which the three enzymes would work coordinately allowing to maintain a sustained glyoxal oxidase activity. In the course of its catalytic cycle, glyoxal oxidase alternates between two functional and interconvertible reduced and oxidized forms resulting from a two-electron transfer process. However, glyoxal oxidase can also undergo an one-electron reduction to a catalytically inactive form lacking the active site free radical. Horseradish peroxidase could use glyoxal oxidase-generated H<SUB>2</SUB>O<SUB>2</SUB> to oxidize Mn(II) to Mn(III) which, in turn, would reoxidize and reactivate the inactive form of glyoxal oxidase. Catalase would remove the excess of H<SUB>2</SUB>O<SUB>2</SUB> generated during the reaction. In spite of the improvement achieved using the three-enzyme system, glyoxal oxidase inactivation still occurred, which resulted in low substrate conversions. Possible causes of inactivation, including end-product inhibition, are discussed.</P>