Jin Kyeong Sik, Rho Yecheol, Kim Jehan, Kim Heesoo, Kim Ik Jung, Ree Moonhor
Department of Chemistry, National Research Laboratory for Polymer Synthesis and Physics, Pohang Accelerator Laboratory, Pohang University of Science and Technology (Postech), Pohang 790-784, Republic of Korea.
J Phys Chem B. 2008 Dec 11;112(49):15821-7. doi: 10.1021/jp805940d.
Structural characteristics of various conformational states of porcine pepsin in solution under different pH conditions were investigated in terms of size and shape by small-angle X-ray scattering (SAXS). Low-resolution structural models of porcine pepsin were reconstructed from SAXS data, which were made inside the search volume of maximum dimension (Dmax), calculated from the pair distance distribution function p(r). The reconstructed structural models were obtained without imposing any restrictions on the symmetry or anisometry of the pepsin molecule. Under conditions emulating those for physiological activity of the enzyme, the reconstructed structural models exhibited a more extended C-terminal domain compared to the crystal structure. The differences between the solution and crystal structures of pepsin can be explained by inherent conformations of the flexible subdomain in the C-terminal domain under the solution pH conditions. Under mild acidic conditions where the enzyme is inactive, the reconstructed structural models revealed a compact globular conformation similar in overall shape to the crystal structure. These results indicate that the changes in fluorescence and circular dichroism curves observed under acidic conditions could also arise from the inherent conformation of the flexible subdomain, which has a tendency to roll into a sphere in the overall structure, but without affecting the stability of internal structure. Furthermore, the conformational changes in the subdomain might explain the inactivity of pepsin under mildly acidic conditions. Finally, compared to neutral denaturing conditions, pepsin under alkaline denaturing conditions had a larger expanded vertical conformation in the reconstructed model, as a consequence of alkaline denaturation of the N-terminal domain and a fully extended conformation of the C-terminal domain. The structural evidence presented here may have important implications for understanding the relationship between the structure of porcine pepsin and enzymatic function.
通过小角X射线散射（SAXS）从尺寸和形状方面研究了不同pH条件下溶液中猪胃蛋白酶各种构象状态的结构特征。根据从对距离分布函数p(r)计算出的最大尺寸（Dmax）的搜索体积内得到的SAXS数据，重建了猪胃蛋白酶的低分辨率结构模型。重建的结构模型是在不对胃蛋白酶分子的对称性或各向异性施加任何限制的情况下获得的。在模拟该酶生理活性的条件下，与晶体结构相比，重建的结构模型显示出C末端结构域更加伸展。胃蛋白酶溶液结构和晶体结构之间的差异可以通过溶液pH条件下C末端结构域中柔性亚结构域的固有构象来解释。在该酶无活性的轻度酸性条件下，重建的结构模型显示出一种紧凑的球状构象，其整体形状与晶体结构相似。这些结果表明，在酸性条件下观察到的荧光和圆二色性曲线的变化也可能源于柔性亚结构域的固有构象，该亚结构域在整体结构中有卷成球体的趋势，但不影响内部结构的稳定性。此外，亚结构域的构象变化可能解释了胃蛋白酶在轻度酸性条件下的无活性。最后，与中性变性条件相比，在碱性变性条件下的胃蛋白酶在重建模型中具有更大的垂直伸展构象，这是由于N末端结构域的碱性变性和C末端结构域的完全伸展构象所致。这里提出的结构证据可能对理解猪胃蛋白酶的结构与酶功能之间的关系具有重要意义。
