Bartunik H D, Summers L J, Bartsch H H
Max-Planck-Society, Research Unit for Structural Molecular Biology, Hamburg, West Germany.
J Mol Biol. 1989 Dec 20;210(4):813-28. doi: 10.1016/0022-2836(89)90110-1.
The crystal structure of bovine pancreatic beta-trypsin (BPT) has been determined from a novel orthorhombic crystal form which contains substantially more solvent (filling 57% of the volume of the unit cell) than previously determined orthorhombic (44%) and trigonal (37%) BPT structures. The native and benzamidine-inhibited crystal structures of BPT in ammonium sulphate at pH 5.3 have been determined for the new form by molecular replacement techniques. The structures have been refined at 1.5 A resolution with final R-values of 16.7% and 16.9%, respectively. Comparison with the previously refined old orthorhombic forms shows that the overall conformation of the protein backbone is highly conserved. A great number of previously undefined side-chains have been located in density. At the C terminus an extra ion pair involving lysines 87 and 107 has been revealed. A far more detailed picture of the ordered solvent structure has been derived. Thirty water clusters have been identified. A large water network extends from the calcium binding site to the activation area and the autolysis loop. There is evidence for a water channel reaching from the depth of the specificity pocket to the nearby protein surface which might be involved in the displacement of water molecules upon substrate binding. A sulphate anion which forms hydrogen bonds to the active site residues His57, Ser195 and Gly193 was for the first time positioned in clearly defined electron density. Interaction with the sulphate ion may explain the increase in the pKa value of His57 at high sulphate concentrations which was observed by nuclear magnetic resonance studies of a bacterial serine protease both in crystalline form and in solution. Thus, a His-Ser hydrogen bond will not exist in solvents containing sulphate at low pH (up to at least 6.8) where the imidazole of His57 is protonated. The new crystal form is of considerable interest for substrate binding studies. Wide solvent channels should allow diffusion of large substrates (comparable in size to, e.g. pancreatic trypsin inhibitor) into the enzyme crystal. The active site is accessible; intermolecular contact areas are further remote from the active site than in the old orthorhombic form.
牛胰β-胰蛋白酶（BPT）的晶体结构是通过一种新型正交晶型确定的，该晶型所含溶剂比先前确定的正交晶型（填充晶胞体积的44%）和三角晶型（填充晶胞体积的37%）要多得多（填充晶胞体积的57%）。通过分子置换技术确定了pH 5.3的硫酸铵中BPT的天然晶体结构和苯甲脒抑制的晶体结构。这些结构已在1.5 Å分辨率下进行了精修，最终R值分别为16.7%和16.9%。与先前精修的旧正交晶型相比，蛋白质主链的整体构象高度保守。大量先前未确定的侧链已在电子密度图中定位。在C末端，发现了一个涉及赖氨酸87和107的额外离子对。得到了更详细的有序溶剂结构图像。已鉴定出30个水簇。一个大的水网络从钙结合位点延伸到激活区域和自溶环。有证据表明存在一条从特异性口袋深处通向附近蛋白质表面的水通道，该通道可能在底物结合时参与水分子的置换。首次在清晰定义的电子密度中定位了一个与活性位点残基His57、Ser195和Gly193形成氢键的硫酸根阴离子。与硫酸根离子的相互作用可能解释了在高硫酸根浓度下His57的pKa值增加的现象，这一现象在细菌丝氨酸蛋白酶的晶体形式和溶液中的核磁共振研究中均有观察到。因此，在低pH（至少高达6.8）且含有硫酸根的溶剂中，His57的咪唑被质子化时，His-Ser氢键将不存在。这种新的晶型对于底物结合研究具有相当大的意义。宽阔的溶剂通道应允许大底物（例如与胰腺胰蛋白酶抑制剂大小相当）扩散到酶晶体中。活性位点是可及的；分子间接触区域比旧的正交晶型离活性位点更远。
