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 reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol caused a drastic decrease in enzymatic activity, whereas the sphingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the uncharged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards erythrocyte membranes.
Tomita
M
M
Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Ueda
Y
Y
Tamura
H
H
Taguchi
R
R
Ikezawa
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D004229:Dithiothreitol; D004910:Erythrocyte Membrane / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D013108:Sphingomyelin Phosphodiesterase; D004229:Dithiothreitol