南极磷虾胰蛋白酶的结构分析及适冷性机制研究

本文基于酶的纯化鉴定、动力学分析、序列比对和结构预测，对南极磷虾胰蛋白酶的结构及适冷性进行研究，通过与其他几种适冷、中温胰蛋白酶进行差异性分析，探讨南极磷虾胰蛋白酶等适冷酶在催化反应过程中应对低温环境的机制。结果显示适冷胰蛋白酶和中温胰蛋白酶的催化中心严格保守，遵循一致的催化机制；但南极磷虾胰蛋白酶等适冷酶为了适应低温环境，各级结构都表现出一定的应对机制：疏水性残基含量偏高、带正负电荷残基含量偏低，分子内部疏水作用增强、盐桥减少，这些因素致使适冷胰蛋白酶蛋白质分子内的相互作用减弱，分子柔性增强。适冷胰蛋白酶具有更高比例的呈松散状的无规卷曲结构，一定程度提高了其柔性；底物专一性口袋周围空间位阻更小，底物更容易接近活性中心，这也是加快催化反应的关键因素。
The molecular characterization of trypsin purified from Euphausia superba (Dana, 1852) was investigated in this research. Aspects such as thermodynamic activation parameters, sequence alignment, and molecular model allowed an in-depth understanding of its activity at low temperatures. Conserved residues were compared between cold-adapted trypsin and its warm-adapted counterparts, and the results showed that their active cores were almost identical. Strategies adopted by trypsin for molecular adaptation to low temperatures may be as follows. A higher proportion of hydrophobic residues and lower proportion of charged residues in the enzyme probably diminish the number of intramolecular interactions, resulting in improved structural flexibility. Increased number of loose random coils may also contribute to improve structural flexibility. Reduced steric hindrance is also a key factor that allows the substrate to easily access the active site, thus promoting the catalytic reaction.