Glu-53 of Bacillus cereus sphingomyelinase acts as an indispensable ligand of Mg2+ essential for catalytic activity.
Bacillus cereus sphingomyelinase (SMase) is an extracellular hemolysin classified into a group of Mg(2+)-dependent neutral SMases (nSMase). Sequence comparison of bacterial and eukaryotic Mg(2+)-dependent nSMases has shown that several amino acid residues, including Glu-53 of B. cereus SMase, are conserved, suggesting a catalytic mechanism common to these enzymes. Mutational analysis has revealed that hemolytic and SM-hydrolyzing activities are abolished by E53A and E53Q mutations. Only the E53D mutant enzyme partially retains these activities, however, a significant decrease in the apparent k(cat)/K(m) for SM hydrolysis is observed by this mutation. Mg(2+) activates the wild-type enzyme in a two-step manner, i.e., at least two binding sites for Mg(2+), high- and low-affinity, are present on the enzyme. The binding affinity of essential Mg(2+) for the high-affinity site is decreased by the mutation. In addition, the binding affinities of Mn(2+) and Co(2+) (substitutes for Mg(2+)) are also decreased. On the contrary, the inhibitory effects of Ca(2+), Cu(2+), and Zn(2+) on SM-hydrolyzing activity are not influenced by the mutation. The results indicate that Glu-53 of B. cereus SMase acts as a ligand for Mg(2+) and is involved in the high-affinity Mg(2+)-binding site, which is independent of the binding site for inhibitory metals.
Obama
Takashi
T
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan.
Kan
Yukie
Y
Ikezawa
Hiroh
H
Imagawa
Masayoshi
M
Tsukamoto
Kikuo
K
D000595:Amino Acid Sequence; D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D010446:Peptide Fragments; D005973:Glutamine; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium