Bioenergy Area, Energy Unit, Tecnalia Research and Innovation, Parque Tecnológico de San Sebastián, Mikeletegi Pasealekua 2, E-20009 San Sebastián, Spain.
Enzyme Microb Technol. 2012 Feb 10;50(2):143-50. doi: 10.1016/j.enzmictec.2011.11.007. Epub 2011 Dec 8.
Glyoxal oxidase of P. chrysosporium is a radical copper oxidase that catalyzes oxidation of aldehydes to carboxylic acids coupled to dioxygen reduction to H(2)O(2). 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(2)O(2)) 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(2)O(2) 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(2)O(2) 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.
白腐真菌的乙二醛氧化酶是一种活性铜氧化酶，可催化醛氧化为羧酸，同时将氧还原为 H(2)O(2)。除了已知的底物外，甘油也被发现是乙二醛氧化酶的底物。在酶转化过程中，乙二醛氧化酶会发生可逆失活，可能是由于活性部位自由基的丢失所致，从而导致酶活性短暂和无法检测到的底物转化。通过在包含两种额外酶（辣根过氧化物酶和过氧化氢酶）以及氧化还原化学激活剂（如 Mn(III)（或 Mn(II)+H(2)O(2)）或六氯合铱酸盐）的情况下，酶活性可以得到延长。使用这种三酶系统，甘油可以在两步反应中转化为甘油酸，中间产物为甘油醛。提出了一种可能的操作机制，其中三种酶将协同工作，以保持持续的乙二醛氧化酶活性。在其催化循环中，乙二醛氧化酶通过两个电子转移过程在两种功能和可相互转化的还原和氧化形式之间交替。然而，乙二醛氧化酶也可以经历单电子还原，形成缺乏活性部位自由基的催化失活形式。辣根过氧化物酶可以利用乙二醛氧化酶产生的 H(2)O(2)将 Mn(II)氧化为 Mn(III)，后者反过来又会将失活形式的乙二醛氧化酶重新氧化并重新激活。过氧化氢酶会去除反应过程中产生的多余的 H(2)O(2)。尽管使用三酶系统可以提高效率，但乙二醛氧化酶的失活仍然会发生，从而导致底物转化率低。讨论了失活的可能原因，包括终产物抑制。
