Study on the Stabilization of Enzyme against Heat and Organic Solvents

酶对热和有机溶剂的稳定性研究

• 批准号: 13836004
• 负责人: HACHIMORI Akira
• 金额: $ 2.3万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13836004/
• 关键词: Escherichia coli; inorganic pyrophosphatse; thermostability; stability for organic solvents; site directed mutagenesis; Bacillus subtilis; thermophilic bacterium PS-3; 部位特異的変異法; 酵素; タンパク; 耐熱性

We found the following results through this study.1.We found that the roles of two lysyl residues among the common sequence KKR (295K and 296K in inorganic pyrophosphatase from Bacillus subtilis) at the C-terminal region in Family 2 inorganic pyrophosphates (PPase) is the recognition site for the substrate. The_arginyl residue (297R in Bacillus subtilis PPase) is important for maintaining the structure for the pass to let the substrate go through.2.Family 2 PPase is composed of two identical subunits, and each subunits contains 3 molecules of manganese ions.3.B. subtilis PPase subunit is composed from 2 domains (N-domain from 1 to 187 and C-domain from 193 to 323), which are connected by the hinge region composed of 6 amino acid residues. Among these 6 resides, 189G is especially important to keep the enzymatically active structure.4.The prolyl residues on the_surface region of the Family 1 PPase from Thermophilic bacterium PS-3 have no role for the heat stability of enzyme. However, the prolyl residues in the interior region are important for maintain the conformation of enzyme. Especially Pro-72 is very important for maintaining the hexamer, which is absolutely necessary for the heat stabilization of enzyme.5.We succeeded in obtaining the variant F40K, Y57I, P74I, Q80L, S111E fo Family 1 PPase from E. coli, and we are now under investigation of their property to obtain the information about the heat stabilization and organic solvent stabilization.

通过本研究，我们得到了以下结果：1.我们发现KKR(枯草芽孢杆菌无机焦磷酸酶的295K和296K)共同序列中的两个赖氨酸残基在PPase的C-末端区域的作用是底物的识别位点。枯草杆菌PPase家族2由2个相同的亚基组成，每个亚基含有3个锰离子分子。3.枯草杆菌PPase亚基由2个结构域(N结构域1~187和C结构域193~323)组成，由6个氨基酸残基组成的铰链区相连。在这6个残基中，189G对保持酶的活性结构尤为重要。4.嗜热细菌PS-3的PPase家族1表面的脯氨酸残基对酶的热稳定性没有影响。然而，内部区域的脯氨基残基对于维持酶的构象是重要的。特别是Pro-72对于维持六聚体是非常重要的，而六聚体对于酶的热稳定是必不可少的。5.我们成功地从大肠杆菌中获得了PPase家族1的变异体F40K、Y57I、P74I、Q80L、S111E，目前我们正在对它们的性质进行研究，以获得有关热稳定和有机溶剂稳定的信息。

项目成果

Nishiyama, T., Hachimori, A., Uchiumi, T., Nakashima, N: "Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes"Nucleic Acids Res.. 31. 2434-2442 (2003)

Nishiyama, T.、Hachimori, A.、Uchiumi, T.、Nakashima, N：“负责与核糖体结合的 Plautia stali 肠病毒内部核糖体进入位点的结构元素”核酸研究 31. 2434-2442（

Shizawa, N., Uchiumi, T., Taguchi, J., Kisseleva, N.A., Baykov, A.A., Lahti, R., Hachimori, A.: "Direct mutagenesis studies of the C-terminal fingerprint region of Bacillus subtilis pyrophosphatase"Eur.J.Biochem.. 268. 1-4 (2001)

Shizawa, N.、Uchiumi, T.、Taguchi, J.、Kisseleva, N.A.、Baykov, A.A.、Lahti, R.、Hachimori, A.：“枯草芽孢杆菌焦磷酸酶 C 端指纹区的直接诱变研究”Eur

Hachimori, A., Baykov, A.A., Lapti, R: "Quaternary structure and metal ion requirement of family 2 pyrophosphatase from B.subtilis, S.gordonii and S.mutans"J.Biol.Chem.. 276. 24511-24518 (2001)

Hachimori, A.、Baykov, A.A.、Lapti, R：“来自 B.subtilis、S.gordonii 和 S.mutans 的家族 2 焦磷酸酶的四级结构和金属离子要求”J.Biol.Chem.. 276. 24511-24518（

Shizawa, N., Uchiumi, T., Hachimori, A.: "Direct mutagenesis studies of the C-terminal fingerprint region of Bacillus subtilis pyrophosphatase"Eur.J.Biochem.. 268. 1-4 (2001)

Shizawa, N.、Uchiumi, T.、Hachimori, A.：“枯草芽孢杆菌焦磷酸酶 C 末端指纹区的直接诱变研究”Eur.J.Biochem.. 268. 1-4 (2001)

Nomura, T., Hachimori, A., Uchiumi, T.et al.: "A point mutation in ribosomalprotein L7/L12 reduce its ability to form a compact dimmer structureand to assemble into GTPase center"Biochemistry. 42. 4691-4698 (2003)

Nomura, T.、Hachimori, A.、Uchiumi, T.等人：“核糖体蛋白 L7/L12 的点突变降低了其形成紧凑二聚体结构和组装成 GTP 酶中心的能力”生物化学。