Chu N M, Chao Y, Bi R C
Department of Protein Engineering, Institute of Biophysics, Academia Sinica, Beijing, PRC.
Protein Eng. 1995 Mar;8(3):211-5. doi: 10.1093/protein/8.3.211.
Using enzyme prepared by the DNA recombination technique, subtilisin E from Bacillus subtilis was crystallized in space group P2(1)2(1)2(1) with two molecules in an asymmetric unit. The crystal structure of PMSF-inhibited subtilisin E was solved by molecular replacement followed by refinement with the X-PLOR program. This resulted in the 2.0 A structure of subtilisin E with an R-factor of 0.191 for 8-2 A data and r.m.s. deviations from ideal values of 0.021 A and 2.294 for bond lengths and bond angles respectively. The PMSF group covalently bound to Ser221 appeared very clearly in the electron density map. Except for the active site disturbed by PMSF binding, the structural features of subtilisin E are almost the same as in other subtilisins. The calcium-binding sites are different in detail in the two independent molecules of subtilisin E. Based on the structure, the remarkably enhanced heat stability of mutant N118S of subtilisin E is discussed. It is very likely that there is an additional water molecule in the mutant structure, which is hydrogen bonded to side chains of Ser118 and its neighbouring residues Lys27 and Asp120.
利用DNA重组技术制备的酶，来自枯草芽孢杆菌的枯草杆菌蛋白酶E在空间群P2(1)2(1)2(1)中结晶，一个不对称单位中有两个分子。通过分子置换法，随后用X-PLOR程序进行精修，解析了苯甲基磺酰氟（PMSF）抑制的枯草杆菌蛋白酶E的晶体结构。这得到了枯草杆菌蛋白酶E的2.0 Å结构，对于8 - 2 Å数据，R因子为0.191，键长和键角与理想值的均方根偏差分别为0.021 Å和2.294。与Ser221共价结合的PMSF基团在电子密度图中非常清晰地显现出来。除了被PMSF结合干扰的活性位点外，枯草杆菌蛋白酶E的结构特征与其他枯草杆菌蛋白酶几乎相同。在枯草杆菌蛋白酶E的两个独立分子中，钙结合位点在细节上有所不同。基于该结构，讨论了枯草杆菌蛋白酶E突变体N118S显著增强的热稳定性。很可能在突变体结构中有一个额外的水分子，它与Ser118及其相邻残基Lys27和Asp120的侧链形成氢键。
