Mg 2+ binding and catalytic function of Sphingomyelinase from Bacillus cereus

Shinobu Fujii, Bunpei Inoue, Hiroki Yamamoto, Kenji Ogata, Tomohiro Shinki, Seiji Inoue, Masahiro Tomita, Hiroomi Tamura, Kikuo Tsukamoto, Hiroh Ikezawa, Kiyoshi Ikeda

Research output: Contribution to journalArticle

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Abstract

The modes of Mg 2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg 2+ with low and high affinities. The effects of Mg 2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg 2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg 2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg 2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/K(m) for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of k(cat) for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.

Original languageEnglish
Pages (from-to)1178-1187
Number of pages10
JournalJournal of Biochemistry
Volume124
Issue number6
Publication statusPublished - 1998

Fingerprint

Bacillus cereus
Sphingomyelin Phosphodiesterase
Denaturation
Deoxyribonuclease I
Octoxynol
Enzymes
Catalysis
Substrates
Enzyme Stability
Kinetic parameters
Urea
Hydrolysis
Fluorescence
Binding Sites
Molecules

Keywords

  • Catalytic mechanism
  • Denaturation
  • Enzyme kinetics
  • Mg binding
  • Sphingomyelinase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Fujii, S., Inoue, B., Yamamoto, H., Ogata, K., Shinki, T., Inoue, S., ... Ikeda, K. (1998). Mg 2+ binding and catalytic function of Sphingomyelinase from Bacillus cereus. Journal of Biochemistry, 124(6), 1178-1187.

Mg 2+ binding and catalytic function of Sphingomyelinase from Bacillus cereus. / Fujii, Shinobu; Inoue, Bunpei; Yamamoto, Hiroki; Ogata, Kenji; Shinki, Tomohiro; Inoue, Seiji; Tomita, Masahiro; Tamura, Hiroomi; Tsukamoto, Kikuo; Ikezawa, Hiroh; Ikeda, Kiyoshi.

In: Journal of Biochemistry, Vol. 124, No. 6, 1998, p. 1178-1187.

Research output: Contribution to journalArticle

Fujii, S, Inoue, B, Yamamoto, H, Ogata, K, Shinki, T, Inoue, S, Tomita, M, Tamura, H, Tsukamoto, K, Ikezawa, H & Ikeda, K 1998, 'Mg 2+ binding and catalytic function of Sphingomyelinase from Bacillus cereus', Journal of Biochemistry, vol. 124, no. 6, pp. 1178-1187.
Fujii S, Inoue B, Yamamoto H, Ogata K, Shinki T, Inoue S et al. Mg 2+ binding and catalytic function of Sphingomyelinase from Bacillus cereus. Journal of Biochemistry. 1998;124(6):1178-1187.
Fujii, Shinobu ; Inoue, Bunpei ; Yamamoto, Hiroki ; Ogata, Kenji ; Shinki, Tomohiro ; Inoue, Seiji ; Tomita, Masahiro ; Tamura, Hiroomi ; Tsukamoto, Kikuo ; Ikezawa, Hiroh ; Ikeda, Kiyoshi. / Mg 2+ binding and catalytic function of Sphingomyelinase from Bacillus cereus. In: Journal of Biochemistry. 1998 ; Vol. 124, No. 6. pp. 1178-1187.
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abstract = "The modes of Mg 2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg 2+ with low and high affinities. The effects of Mg 2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg 2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg 2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg 2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/K(m) for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of k(cat) for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.",
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T1 - Mg 2+ binding and catalytic function of Sphingomyelinase from Bacillus cereus

AU - Fujii, Shinobu

AU - Inoue, Bunpei

AU - Yamamoto, Hiroki

AU - Ogata, Kenji

AU - Shinki, Tomohiro

AU - Inoue, Seiji

AU - Tomita, Masahiro

AU - Tamura, Hiroomi

AU - Tsukamoto, Kikuo

AU - Ikezawa, Hiroh

AU - Ikeda, Kiyoshi

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N2 - The modes of Mg 2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg 2+ with low and high affinities. The effects of Mg 2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg 2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg 2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg 2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/K(m) for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of k(cat) for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.

AB - The modes of Mg 2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg 2+ with low and high affinities. The effects of Mg 2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg 2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg 2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg 2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/K(m) for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of k(cat) for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.

KW - Catalytic mechanism

KW - Denaturation

KW - Enzyme kinetics

KW - Mg binding

KW - Sphingomyelinase

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