Roles of Asp126 and Asp156 in the enzyme function of sphingomyelinase from Bacillus cereus

Shinobu Fujii, Kenji Ogata, Bunpei Inoue, Seiji Inoue, Masahiro Murakami, Seiji Iwama, Shigeo Katsumura, Masahiro Tomita, Hiro Omi Tamura, Kikuo Tsukamoto, Hiroh Ikezawa, Kiyoshi Ikeda

研究成果: Article査読

7 被引用数 (Scopus)


To elucidate the roles of conserved Asp residues of Bacillus cereus sphingomyelinase (SMase) in the kinetic and binding properties of the enzyme toward various substrates and Mg2+, the kinetic data on mutant SMases (D126G and D156G) were compared with those of wild type (WT) enzyme. The stereoselectivity of the enzyme in the hydrolysis of monodispersed short-chain sphingomyelin (SM) analogs and the binding of Mg2+ to the enzyme were not affected by the replacement of Asp126 or Asp156. The pH-dependence curves of kinetic parameters (1/K(m) and k(cat)) for D156G-catalyzed hydrolysis of micellar SM mixed with Triton X-100 (1:10) and of micellar 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP) were similar in shape to those for WT enzyme-catalyzed hydrolysis. On the other hand, the curves for D126G lacked the transition observed for D156G and WT enzymes. Comparison of the values and the shape of pH-dependence curves of kinetic parameters indicated that Asp126 of WT SMase enhances the enzyme's catalytic activity toward both substrates and its binding of HNP but not SM. The deprotonation of Asp126 enhances the substrate binding and slightly suppresses the catalytic activity toward both substrates. Asp156 of WT SMase acts to decrease the binding of both substrates and the catalytic activity to HNP but not SM. From the present study and the predicted three-dimensional structure of B. cereus SMase, Asp126 was thought to be located close to the active site, and its ionization was shown to affect the catalytic activity and substrate binding.

ジャーナルJournal of biochemistry
出版ステータスPublished - 1999

ASJC Scopus subject areas

  • 生化学
  • 分子生物学


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