Mg2+ binding and catalytic function of sphingomyelinase from Bacillus cereus

The modes of Mg2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg2+ with low and high affinities. The effects of Mg2+ binding on the enzymatic activity and stru ctural stability of the enzyme molecule were also studied. The results indi cated that the binding of Mg2+ to the low-affinity site was essential for t he catalysis, but was independent of the substrate binding to the enzyme. I t was also indicated that the alkaline denaturation of the enzyme was partl y prevented by the Mg2+ binding, whereas no significant protective effect w as observed against the denaturation by urea. The pH dependence of the kine tic parameters for the hydrolysis of micellar HNP and mixed micellar SM wit h Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in th e presence of a large amount of Mg2+ to saturate both the low- and high-aff inity sites. The pH dependence curves of the logarithm of 1/K-m for these t wo kinds of substrates were similar in shape to each other, and showed a si ngle transition. On the other hand, the shapes of the pH dependence curves of the logarithm of k(cat), for these two kinds of substrates were differen t from each other. The pH dependence curve for micellar HNP showed three tr ansitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectiv ely. On the other hand, the curve for mixed micellar SM with Triton X-100 s howed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present r esults and the three-dimensional structure of bovine pancreatic DNase I, wh ich has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.