Study on the Stabilization of Enzyme against Heat and Organic Solvents

通过本研究，我们发现：1.在第二家族无机焦磷酸酶（PPase）的C-末端区域的共同序列KKR（枯草芽孢杆菌无机焦磷酸酶中的295 K和296 K）中，两个赖氨酰残基的作用是底物的识别位点。枯草杆菌PPase中的297 R残基对维持底物通过通道的结构至关重要。2.家族2 PPase由两个相同的亚基组成，每个亚基含有3个锰离子分子。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，这2个结构域通过由6个氨基酸残基组成的铰链区连接。4.嗜热菌PS-3家族1 PPase的表面脯氨酰残基对酶的热稳定性没有影响。然而，脯氨酰残基的内部区域是重要的维持酶的构象。Pro-72对维持酶的热稳定性至关重要。5.成功地从大肠杆菌中获得了家族1 PPase的变异体F40 K、Y 57 I、P74 I、Q80 L、S111 E。我们正在研究它们的性质，以获得有关热稳定性和有机溶剂稳定性的信息。

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.