STUDIES ON THE ACTIVE-SITES OF BACILLUS-CEREUS SPHINGOMYELINASE SUBSTITUTION OF SOME AMINO-ACIDS BY SITE-DIRECTED MUTAGENESIS

Chemical modifications suggested that acidic amino acids such as aspar tic and glutamic acids are involved in the active sites of Bacillus ce reus sphingomyelinase. Among aspartic acid residues in the conserved r egions of this enzyme, Asp-126, Asp-156, Asp-233 and Asp-295 were conv erted to glycine by site-directed mutagenesis. According to prediction on structural similarity to pancreatic DNase I, His-151 and His-296 w ere also converted to alanine. The Asp and His mutants, D126G, D156G, D233G, D295G, H151A and H296A, were produced in Bacillus brevis 47, a protein-hyperproducing strain. The catalytic activities of D295G, H151 A and H296A were completely abolished, and sphingomyelin-hydrolyzing a ctivity of D126G or D156G was reduced by more than 50%. The activity o f D126G toward p-NPPC was comparable to that of the wild-type, while D 156G catalyzed the hydrolysis of HNP and p-NPPC more efficiently than the wild-type. Hemolytic activities of the mutants were parallel to th eir sphingomyelin-hydrolyzing activities.