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J. Biochem, 2003, Vol. 133, No. 3 279-286
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

Glu-53 of Bacillus cereus Sphingomyelinase Acts as an Indispensable Ligand of Mg2+ Essential for Catalytic Activity

Takashi Obama, Yukie Kan, Hiroh Ikezawa, Masayoshi Imagawa and Kikuo Tsukamoto+,

Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603

Bacillus cereus sphingomyelinase (SMase) is an extracellular hemolysin classified into a group of Mg2+-dependent neutral SMases (nSMase). Sequence comparison of bacterial and eukaryotic Mg2+-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 kcat/Km for SM hydrolysis is observed by this mutation. Mg2+ activates the wild-type enzyme in a two-step manner, i.e., at least two binding sites for Mg2+, high- and low-affinity, are present on the enzyme. The binding affinity of essential Mg2+ for the high-affinity site is decreased by the mutation. In addition, the binding affinities of Mn2+ and Co2+ (substitutes for Mg2+) are also decreased. On the contrary, the inhibitory effects of Ca2+, Cu2+, and Zn2+ 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 Mg2+ and is involved in the high-affinity Mg2+-binding site, which is independent of the binding site for inhibitory metals.

+ To whom correspondence should be addressed. Tel: +81-52-836-3458, Fax: +81-52-836-3457, E-mail: kikuot{at}phar.nagoya-cu.ac.jp


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