Using D- and L-Amino Acid Oxidases to Generate the Imino Acid Substrate to Measure the Activity of the Novel Rid (Enamine/Imine Deaminase) Class of Enzymes.

This chapter describes a method to assay the activity of reactive intermediate deaminases (Rid), a large family of conserved soluble enzymes, which have been proposed to prevent damages from metabolic intermediates such as the highly reactive and unstable compounds enamines/imines. In this method, the flavin adenine dinucleotide-dependent L- or D-amino acid oxidases generate an imino acid starting from a L- or D- amino acid, respectively. This reaction is coupled to the hydrolysis of the imino acid to the corresponding α-keto acid and ammonium ion catalyzed by a Rid enzyme. The spectrophotometric assay consists of measuring the decrease of the initial rate of formation of the semicarbazone, derived from the spontaneous reaction of the imino acid and semicarbazide, caused by the presence of the Rid enzyme. The set-up and testing of this method imply a preliminary characterization of the ability of the amino acid oxidase to release the imino acid required for the subsequent reactions. To this purpose, the activity of the L- or D-amino acid oxidases with different amino acids can be measured as production of hydrogen peroxide or formation of semicarbazone in parallel assays. The advantages and limitations of this assay of Rid activity are discussed.