Inactivation of Choline Oxidase by Irreversible Inhibitors or Storage Conditions

Choline oxidase from Arthrobacter globiformis is a flavin-dependent enzyme that catalyzes the oxidation of choline to betaine aldehyde through two sequential hydride-transfer steps. The study of this enzyme is of importance to the understanding of glycine betaine biosynthesis found in pathogenic bacterial or economic relevant crop plants as a response to temperature and salt stress in adverse environment. In this study, chemical modification of choline oxidase using two irreversible inhibitors, tetranitromethane and phenylhydrazine, was performed in order to gain insights into the active site structure of the enzyme. Choline oxidase can also be inactivated irreversibly by freezing in 20 mM sodium phosphate and 20 mM sodium pyrophosphate at pH 6 and -20 C. The results showed that enzyme inactivation was due to a localized conformational change associated with the ionization of a group in close proximity to the flavin cofactor and led to a complete lost of catalytic activity. INDEX WORDS: Choline Oxidase, Flavoproteins, Enzyme Inactivation, Chemical Modification, Phenylhydrazine, Tetranitromethane, Mechanism-based Inhibitors, Hysteresis.