Studies on Amino Acid Racemases: I. PARTIAL PURIFICATION AND PROPERTIES OF THE ALANINE RACEMASE FROM LACTOBACILLUS FERMENTI

Abstract

Abstract The alanine racemase from Lactobacillus fermenti has been purified 800-fold with a yield of 8%. The enzyme required pyridoxal phosphate as a cofactor, and the dissociation constant was calculated to be 0.15 µm. A sulfhydryl compound was necessary for maximal activity. Either glutathione or 2-mercaptoethanol was effective. The pH optimum was between pH 9.0 and 9.5. The enzyme was stabilized in its most active form by acetate ion. In the presence of acetate the Km for l-alanine was 10 mm; the Km for d-alanine was 7.3 mm, and the enzyme was inhibited by d-cysteine, d-serine, and d-α-amino-n-butyrate but not the corresponding l isomers. In the absence of acetate the enzyme had a lower specific activity, the Km for l-alanine was 16 mm; the Km for d-alanine was 15 mm, and the enzyme was inhibited by d- and l-cysteine, l-serine, and d- and l-α-amino-n-butyrate. A more detailed study of the recemase inhibition by d-cysteine indicated that with l-alanine as substrate, the slopes of the 1/v versus 1/[S]o plots were increased but the 1/v intercepts did not change. With d-alanine as substrate, the slopes and both the 1/v and 1/[S]o intercepts were altered. The only model of enzymatic behavior which predicted this effect was E ⇌ EL ⇌ FD ⇌ F + D E ⇌ F E + I ⇌ EI where E and F are two forms of the enzyme, D is d-alanine, L is l-alanine, and I is the inhibitor.