Isozymic nature of spore coat-associated alanine racemase of Bacillus subtilis.

Spore coat-associated alanine racemase of Bacillus subtilis, which converts L-alanine to D-alanine, that is, the germinant to the competitive inhibitor, to regulate spore germination for survival of the organism under unfavorable growth conditions, was examined. The dormant spores, L-alanine-initiated germination of which is inhibited by diphenylamine, were used to characterize the enzyme in the native form because of its unextractablility from dormant spores. The presence of isozymes, Enz-I and Enz-II with Km for L-alanine of about 20mM and 50mM and optimum activity at around 40 degrees C and 65 degrees C, respectively, was proposed. The enzymes were selectively used depending on the L-alanine concentration and the temperature. The pH profiles of the activity (optimun at pH 9.0) and the stability (stable between pH 6-11 at 60 degrees C) were similar, but Enz-II was more heat-stable than Enz-I and the denaturation curve demonstrated a two-domain structure for Enz-II. Sensitivity to D-penicillamine, hydroxylamine and HgCl2 was similar between Enz-I and Enz-II, while that to D-cycloserine, L- and D-aminoethylphosphonic acid, monoiodoacetate and N-ethylmaleimide was different; HgCl2 was the most effective inhibitor among these compounds.