Yamane T, Iwasaki A, Suzuki A, Ashida T, Kawata Y
Department of Biotechnology, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi.
J Biochem. 1995 Nov;118(5):882-94. doi: 10.1093/jb/118.5.882.
A trypsin-like serine protease from Streptomyces erythraeus (abbreviated as SET) has been crystallized at pH 7, which is within its active pH range. The crystal structure of SET has been solved by molecular replacement using the atomic model of Streptomyces griseus trypsin (SGT), which is 37% homologous with SET, and refined to the crystallographic R factor of 0.199 for 15,878 reflections with Fo/sigma(F) > 3 between 7 and 1.9 A resolution. The final model of SET contains 1,619 protein atoms and 97 water molecules. No Ca2+ ion is present in SET apparently because (i) the two carboxylate groups from two Glu residues, which bind a Ca2+ ion in bovine trypsin (BT) or SGT, have disappeared; and (ii) a guanidino group from an Arg residue is unfavorably present in the potential binding region. There is an unusual type II beta-turn in which the third residue is Asp instead of Gly. This Asp residue is the only non-Gly residue significantly outside the allowed regions in the Ramachandran map. The three-dimensional structure of SET is essentially the same as those of other trypsins of mammalian origin. The 211 C alpha atoms of SET exhibit an r.m.s. deviation of 1.16 A with equivalent atoms of BT, and the 208 equivalent C alpha atoms between SET and SGT exhibit an r.m.s. deviation of 1.09 A. The large deviations in C alpha positions between SET and BT or between SET and SGT are mainly observed in the first domain. The conformations of the side-chains of the catalytic triad are mutually similar to each other in these three proteases. Each of the chi 1 torsion angles of the three residues is distributed within +/- 5 degrees from each corresponding mean value. The hydrogen bond distances related to the side-chains in the triad coincide fairly well, though the relative disposition of the side-chains differs by 0.1-0.6 A among SET, BT, and SGT. The hydrogen bond network concerned with Ser(195), Asp(189), and water molecules in the substrate binding pocket differs from that in BT or SGT.
来自红链霉菌的一种类胰蛋白酶丝氨酸蛋白酶（简称为SET）已在pH 7条件下结晶，该pH值处于其活性pH范围内。SET的晶体结构已通过分子置换法解析，使用的是灰色链霉菌胰蛋白酶（SGT）的原子模型，SGT与SET有37%的同源性，在7至1.9 Å分辨率下，对15878个F₀/σ(F) > 3的反射进行精修，晶体学R因子达到0.199。SET的最终模型包含1619个蛋白质原子和97个水分子。SET中显然不存在Ca²⁺离子，原因如下：（i）在牛胰蛋白酶（BT）或SGT中结合Ca²⁺离子的两个谷氨酸残基的两个羧基消失了；（ii）在潜在结合区域存在一个来自精氨酸残基的胍基，位置不利。存在一种不寻常的II型β-转角，其中第三个残基是天冬氨酸而非甘氨酸。这个天冬氨酸残基是拉氏图中唯一显著超出允许区域的非甘氨酸残基。SET的三维结构与其他哺乳动物来源的胰蛋白酶基本相同。SET的211个Cα原子与BT的等效原子的均方根偏差为1.16 Å，SET和SGT之间的208个等效Cα原子的均方根偏差为1.09 Å。SET与BT或SET与SGT之间Cα位置的较大偏差主要出现在第一个结构域。在这三种蛋白酶中，催化三联体侧链的构象彼此相似。三个残基的每个χ₁扭转角均分布在各自相应平均值的±5°范围内。尽管SET、BT和SGT之间侧链的相对位置相差0.1 - 0.6 Å，但与三联体侧链相关的氢键距离相当吻合。底物结合口袋中与丝氨酸（195）、天冬氨酸（189）和水分子相关的氢键网络与BT或SGT中的不同。
