Recent Investigations of l- and d-Amino Acid Oxidases

Abstract

Recent research on the flavoprotein amino acid oxidases is reviewed, and several problems relating to the physiological significance of the oxidases and the mechanism of the enzymatic reaction are discussed. The available evidence appears to exclude formation of an α,β-unsaturated intermediate in the reaction, and suggests participation of an imino acid intermediate. Synthetic Δ¹-pyrroline-2-carboxylic acid, the presumed intermediate in the oxidation of proline, was available for direct examination of its possible participation in the amino acid oxidase reaction. Thus, enzymatic synthesis of d-proline from Δ¹-pyrroline-2-carboxylic acid was demonstrated. Synthesis of other d-amino acids from the corresponding α-keto acids and ammonia was also catalyzed by d-amino acid oxidase. Reversibility of the reaction catalyzed by l-amino acid oxidase was also demonstrated. These studies support the belief that an imino acid intermediate is involved in the amino acid oxidation reaction, and make possible new approaches to the study of the mechanism of the enzymatic reactions. Studies on the inhibition of amino acid oxidase by high concentrations of substrate and the effect of oxygen tension are described; a tentative explanation for the results is proposed in which the formation of a semiquinone intermediate is considered. Reactions catalyzed by d-amino acid oxidase involving d-allohydroxyproline and d-pipecolic acid are described. The nonenzymatic formation of pyrrole-2-carboxylic acid from Δ¹-pyrroline-4-hydroxy-2-carboxylic acid (formed enzymatically from d-allohydroxyproline) has provided a highly sensitive method for determining d-amino acid oxidase activity. Crystalline l-amino acid oxidase has been prepared in this laboratory from rattlesnake venom, which contained three separable l-amino acid oxidases. Significant differences between the l-amino acid oxidases of rattlesnake and moccasin venoms are discussed. The physiological significance of d-amino acid oxidase and several possible explanations for its presence in mammalian tissues are considered. It was found that the kidneys of germ-free mice exhibit d-amino acid oxidase activity comparable to that of the kidneys of mice raised under nonsterile conditions. These studies appear to indicate that d-amino acid oxidase activity does not develop adaptively to d-amino acids formed by the bacterial flora.