Irreversible inactivation of the flavoenzyme alcohol oxidase by cyclopropanone

Incubation of the flavoenzyme alcohol oxidase from Candida boidinii with cyclopropanone hydrate in phosphate buffer at pH 7.5 and 20°C leads to a time dependent inactivation of the enzyme which cannot be reversed by removal of the cyclopropanone hydrate. The rate of inactivation is reduced in the presence of substrate, product, or glutathione, and the inactivation is prevented completely by reduction of the FAD coenzyme prior to addition of the inactivator. Inactivation of the enzyme is accompanied by a change in the absorption spectrum of the enzyme which indicates loss of the oxidized flavin chromophore. Denaturation of the inactivated enzyme under a variety of conditions does not cause reoxidation of the flavin which therefore must be covalently modified by cyclopropanone hydrate. A mechanism for the inactivation is proposed which involves radical intermediates and which suggests a similarity in reaction mechanism between alcohol oxidase and flavoprotein amine oxidases.