d -Alanine Oxidase from Escherichia coli : Participation in the Oxidation of l -Alanine

Abstract

Cell wall-membrane preparations of Escherichia coli , prepared by the ethylenediaminetetraacetic acid-lysozyme method, contain enzymes which catalyze the oxidation of d -alanine and, to a lesser extent, l -alanine into pyruvate and ammonia without the formation of hydrogen peroxide. The kinetic parameters were (i) pH optima of 8.3 to 8.4 for l - and d -alanine and (ii) a K m value of 6.6 ± 0.2 mM for d -alanine. Several coenzymes were without effect when added to the reaction mixture. The participation of d -alanine oxidase in the oxidation of l -alanine was demonstrated. The evidence is based on (i) results of cellular fractionation; (ii) labeling experiments; (iii) inhibition studies with aminooxyacetate and cycloserine; (iv) denaturation experiments; and (v) demonstration of the presence of an active racemase.