THE ROLE OF ACIDIC AMINO-ACID-RESIDUES IN CATALYTIC AND ADSORPTIVE SITES OF BACILLUS-CEREUS SPHINGOMYELINASE

By the modification of acidic amino-acid residues with Woodward's reag ent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate), the activity of sphi ngomyelinase of Bacillus cereus was decreased by 80-90%. Also, the red uction of Cys residues in the sphingomyelinase molecule by dithiothrei tol caused a drastic decrease in enzymatic activity, whereas the sphin gomyelinase activity was not affected by treatment with p-chloromercur ibenzenesulfonic acid. Actually, no inactivation of sphingomyelinase a ctivity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the uncharged amino-acid res idues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibit ion of the specific adsorption of sphingomyelinase toward intact eryth rocyte membranes. However, the extent of inhibition in the enzyme adso rption, 20-50%, was less than that observed in the sphingomyelinase ac tivity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the cat alytic sites and the adsorptive sites. Apparently, the disruption of d isulfide linkage in the sphingomyelinase molecule by dithiothreitol de stabilized its structure, resulting in a drastic decrease in sphingomy elin-hydrolyzing activity and specific adsorption of sphingomyelinase towards erythrocyte membranes.