Sugawara Kanako, Tajima Youichi, Kawashima Ikuo, Tsukimura Takahiro, Saito Seiji, Ohno Kazuki, Iwamoto Kunihiko, Kobayashi Toshihide, Itoh Kohji, Sakuraba Hitoshi
Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
Mol Genet Metab. 2009 Apr;96(4):233-8. doi: 10.1016/j.ymgme.2008.12.017. Epub 2009 Jan 31.
Enzyme enhancement therapy (EET) for Fabry disease involving imino sugars has been developed and attracted interest. It is thought that imino sugars act as pharmacological chaperones for wild-type and mutant alpha-galactosidases (GLAs) in cells, but the mechanisms underlying the molecular interactions between the imino sugars and the enzyme have not been clarified yet. We examined various kinds of imino sugars and found that galactostatin bisulfite (GBS) inhibited GLA in vitro and increased the enzyme activity in cultured Fabry fibroblasts as in the case of 1-deoxygalactonojirimycin (DGJ). Then, we analyzed the molecular interactions between the imino sugars and recombinant human GLA by means of isothermal titration calorimetry and surface plasmon resonance biosensor assays, and first determined the thermodynamic and binding-kinetics parameters of imino sugar and GLA complex formation. The results revealed that DGJ bound to the enzyme more strongly than GBS, the binding of DGJ to the enzyme protein being enthalpy-driven. In the case of GBS, the reaction was mainly enthalpy-driven, but there was a possibility that entropy-driven factors were involved in the binding. Structural analysis in silico revealed that both the chemicals fit into the active-site pocket and undergo hydrogen bonding with residues comprising the active-site pocket including the catalytic ones. The side chain of GBS was oriented towards the entrance of the active-site pocket, and thus it could be in contact with residues comprising the wall of the active-site pocket. Thermodynamic, kinetic and structural studies should provide us with a lot of information for improving EET for Fabry disease.
已开发出用于法布里病的涉及亚氨基糖的酶增强疗法（EET）并引起了关注。据认为，亚氨基糖在细胞中作为野生型和突变型α-半乳糖苷酶（GLA）的药理学伴侣，但亚氨基糖与该酶之间分子相互作用的潜在机制尚未阐明。我们研究了各种亚氨基糖，发现半乳糖抑素亚硫酸氢盐（GBS）在体外抑制GLA，并像1-脱氧半乳糖野尻霉素（DGJ）一样增加培养的法布里成纤维细胞中的酶活性。然后，我们通过等温滴定量热法和表面等离子体共振生物传感器测定法分析了亚氨基糖与重组人GLA之间的分子相互作用，并首次确定了亚氨基糖与GLA复合物形成的热力学和结合动力学参数。结果表明，DGJ与该酶的结合比GBS更强，DGJ与酶蛋白的结合是由焓驱动的。在GBS的情况下，反应主要是由焓驱动的，但有可能熵驱动因素参与了结合。计算机模拟结构分析表明，这两种化学物质都适合进入活性位点口袋，并与包括催化残基在内的构成活性位点口袋的残基形成氢键。GBS的侧链朝向活性位点口袋的入口，因此它可能与构成活性位点口袋壁的残基接触。热力学、动力学和结构研究应为我们改善法布里病的EET提供大量信息。
