Asghari S Mohsen, Khajeh Khosro, Ranjbar Bijan, Sajedi Reza H, Naderi-Manesh Hossein
Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modarres University, P.O. Box 14115-175, Tehran, Iran.
Int J Biol Macromol. 2004 Jun;34(3):173-9. doi: 10.1016/j.ijbiomac.2004.03.006.
Detailed circular dichroism (CD), scattering and quenching studies, 1-anilinonaphthalene-8-sulfonate (ANS) binding, irreversible thermoinactivation, activity measurements and proteolytic digestion of bacterial alpha-amylases have been carried out to elucidate the effect of trifluoroethanol (TFE) on the structure of these enzymes. Under high concentrations of TFE both of the alpha-amylases, a thermostable alpha-amylase from Bacillus licheniformis (BLA) and its mesophilic counterpart from Bacillus amyloliquefaciens (BAA), acquire partially folded state characterized by an enhanced content of the secondary structure (helix) and reduced tertiary structures. According to ANS binding studies, we suggest that the TFE states induced by TFE/water mixture are not the molten globule state in the alpha-amylase folding pathway. In addition, data shows significant reversible aggregation of both enzymes in TFE/water mixtures with concentration between 10 and 60% (v/v). However, reversibility is more in case of BAA. As expected, in the absence of TFE, the thermophilic enzyme compared to mesophilic enzyme, shows a greater resistance to digestion by thermolysin. With respect to fluorescence quenching by acrylamide and potassium iodide, the thermophilic enzyme, BLA, is characterized by higher structural flexibility as compared to the BAA. On the other hand, in the presence of TFE, the enzymes are digested by protease to produce large protein fragments. It is proposed that highly helical secondary structures, acquired by BAA and BLA when dissolved in aqueous TFE, prevent binding and adaptation of the protein substrate at the active site of the protease.
已对细菌α-淀粉酶进行了详细的圆二色性（CD）、散射和猝灭研究、1-苯胺基萘-8-磺酸盐（ANS）结合、不可逆热失活、活性测量及蛋白水解消化，以阐明三氟乙醇（TFE）对这些酶结构的影响。在高浓度TFE条件下，两种α-淀粉酶，即来自地衣芽孢杆菌的耐热α-淀粉酶（BLA）及其来自解淀粉芽孢杆菌的嗜温对应物（BAA），都获得了部分折叠状态，其特征是二级结构（螺旋）含量增加，三级结构减少。根据ANS结合研究，我们认为TFE/水混合物诱导的TFE状态不是α-淀粉酶折叠途径中的熔球态。此外，数据显示在TFE/水混合物浓度为10%至60%（v/v）时，两种酶都有明显的可逆聚集。然而，BAA的可逆性更强。正如预期的那样，在没有TFE的情况下，与嗜温酶相比，嗜热酶对嗜热菌蛋白酶的消化具有更大的抗性。关于丙烯酰胺和碘化钾的荧光猝灭，与BAA相比，嗜热酶BLA具有更高的结构灵活性。另一方面，在TFE存在的情况下，这些酶被蛋白酶消化产生大的蛋白质片段。有人提出，BAA和BLA溶解在TFE水溶液中时获得的高度螺旋二级结构会阻止蛋白质底物在蛋白酶活性位点的结合和适配。
