Evidence For An Alkoxide Intermediate In The Reaction Catalyzed By Alcohol Oxidase
Abstract
Alcohol oxidase from the yeast Candida boidinii is a flavin enzyme that catalyzes the oxidation of primary alcohols to their respective aldehydes using molecular oxygen.
R—CH₂—OH + O₂ ⟶ R—CHO + H₂O₂
The function of alcohol oxidase may involves in the metabolism of methanol as a carbon source. The enzyme is a octamer with subunits of 74,000 that are nonconvalently associated (Sahm and Wagner).
The enzyme contains one tightly bound FAD per subunit (Sahm et al). The native enzyme stabilizes a FAD semiquinone radical, which is catalytically inactive. As many as 30% of FAD of the enzyme is in the radical form (Geissler et al). Alcohol oxidase has a narrow substrate specificity.
Alcohol oxidase belongs to a large family of flavin dependent oxidases. Most members of this family catalyze reactions utilizing carbanion intermediate (Ghisla). A well-studied example is D-amino-acid oxidase. D-Amino-acid oxidase oxidizes D-amino acids and gives imino acids as primary products. Three lines of experimental evidence suggest that amino-acid oxidation may begin by enzyme-mediated abstraction of the hydrogen at C-2 as a proton and the formation of a carbanion (Walsh).
The primary indication for the carbanion intermediates is from the study on ring-substituted phenylglycines. The results indicate that substituents that stabilize carbanionic species increased Vₘₐₓ values, where as substituents that destabilize carbanions decreased Vₘₐₓ.
In contrast, there is evidence suggesting that alcohol oxidase follows a radical mechanism. Cyclopropanol irreversibly inactivates alcohol oxidase as a suicide inhibitor but not other oxidases. Alcohol oxidase is not inhibited by the substrates that form covalent flavin N(5) adducts, which are suicide inhibitors for several flavin oxidases that are known to form carbanion intermediates (Geissler et al). The experiments described in this thesis were designed to obtain evidence for or against this proposed radical mechanism.
Description
Program year: 1989/1990Digitized from print original stored in HDR
Citation
Hsieh, Chang-Tai (1990). Evidence For An Alkoxide Intermediate In The Reaction Catalyzed By Alcohol Oxidase. University Undergraduate Fellow. Available electronically from https : / /hdl .handle .net /1969 .1 /CAPSTONE -DawsonL _1977.