Glu-53 of Bacillus cereus Sphingomyelinase Acts as an Indispensable Ligand of Mg2+ Essential for Catalytic Activity
| Autor: | Masayoshi Imagawa, Kikuo Tsukamoto, Takashi Obama, Hiroh Ikezawa, Yukie Kan |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Cation binding
Stereochemistry Glutamine Molecular Sequence Data Bacillus cereus Sphingomyelin phosphodiesterase Ligands Biochemistry Catalysis Animals Magnesium Amino Acid Sequence Binding site Molecular Biology chemistry.chemical_classification Binding Sites biology Active site General Medicine Ligand (biochemistry) biology.organism_classification Peptide Fragments Sphingomyelin Phosphodiesterase Enzyme Cereus chemistry biology.protein Cattle |
| Zdroj: | Journal of Biochemistry. 133:279-286 |
| ISSN: | 0021-924X |
| DOI: | 10.1093/jb/mvg038 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|