Reactivity of the imino acids formed in the amino acid oxidase reaction

Abstract

The reactivity of the imino acids formed in the D- or L-amino acid oxidase reaction was studied. It was found that: (1) When imino acids reacted with the alpha-amino group of glycine or other amino acids, transimination yielded derivatives less stable to hydrolysis than the parent imino acids. In contrast, when imino acids reacted with the epsilon-amino group of lysine or other primary amines, transimination yielded derivatives more stable to hydrolysis than the parent imino acids. (2) Imino acids react rapidly with hydrazine and semicarbazide, forming stable hydrazones and semicarbazones. At pH 7.7, the rate of reaction of the imino acid analogue of leucine with semicarbazide was 10(4) times greater than that of the corresponding keto acid. The reaction of imino acids with these reagents is rapid enough to permit one to follow spectrophotometrically the amino acid oxidase reaction. Imino acids also reacted with cyanide to yield stable adducts. (3) The rate of hydrolysis of the imino acid analogue of leucine was independent of pH above pH 8.5. At lower pH values, the rate of hydrolysis increased with decreasing pH. At 25 degrees C and in the absence of added amino compounds, this imino acid had a half-life of 22 s at pH 8.5. Its half-life was 9.9 s at pH 7.9.

Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids

“…To assess the ability of plant RidA proteins to hydrolyze the reactive imines produced by Thr dehydratase, we developed a new RidA activity assay based on the rapid reaction of semicarbazide with imines to produce UV-absorbing semicarbazones (Hafner and Wellner, 1979). In this assay, the RidA-mediated hydrolysis of imines produced by Thr dehydratase from Thr or Ser is monitored spectrophotometrically via the ability of hydrolysis to compete with, and thereby reduce, semicarbazone formation ( Figure 3A).…”

Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate β-lyase

“…In the case of L-glutamine, owing to >99% cyclization to a lactam [30], the α-keto acid generated in the L-amino acid oxidase reaction cannot be readily quantitated with added 2,4-dinitrophenylhydrazine in an end-point assay. The product of the reaction of L-amino acid oxidase reaction, however, can be continuously trapped as the semicarbazone adduct if semicarbazide is included in the reaction mixture [31]. Typical ε 248nm values for α-keto acid semicarbazones are ~10,000 M −1 cm −1 [32].…”

Spectrophotometric Assays for l-Lysine α-Oxidase and γ-Glutamylamine Cyclotransferase

“…L-Amino acid oxidase catalyzes the oxidation of a large number of L-amino acids with the concomitant formation of H 2 O 2 and ammonia. Although the final product of the reaction catalyzed by this enzyme is usually an ␣-keto acid, the initial enzyme-catalyzed product formed by transfer of two electrons from the substrate to O 2 is an ␣-imino acid (3,4). The ␣-imino acid is unstable and is rapidly (within seconds) hydrolyzed nonenzymatically via the corresponding carbinolamine to an ␣-keto acid and ammonia (4).…”

“…Although the final product of the reaction catalyzed by this enzyme is usually an ␣-keto acid, the initial enzyme-catalyzed product formed by transfer of two electrons from the substrate to O 2 is an ␣-imino acid (3,4). The ␣-imino acid is unstable and is rapidly (within seconds) hydrolyzed nonenzymatically via the corresponding carbinolamine to an ␣-keto acid and ammonia (4). Despite its short half-life, the ␣-imino acid product of the L-amino acid oxidase reaction can be effectively trapped as the corresponding ␣-keto acid semicarbazone in the presence of semicarbazide with the elimination of ammonia (4).…”

“…The ␣-imino acid is unstable and is rapidly (within seconds) hydrolyzed nonenzymatically via the corresponding carbinolamine to an ␣-keto acid and ammonia (4). Despite its short half-life, the ␣-imino acid product of the L-amino acid oxidase reaction can be effectively trapped as the corresponding ␣-keto acid semicarbazone in the presence of semicarbazide with the elimination of ammonia (4). At near neutral pH, the rate of the reaction of semicarbazide with the ␣-imino acid generated from L-leucine is about 10 4 times greater than that obtained with the corresponding ␣-keto acid (4).…”