Heydenreich A, Koellner G, Choe H W, Cordes F, Kisker C, Schindelin H, Adamiak R, Hahn U, Saenger W
Institut für Kristallographie, Freie Universität Berlin, Germany.
Eur J Biochem. 1993 Dec 15;218(3):1005-12. doi: 10.1111/j.1432-1033.1993.tb18459.x.
The crystal structure of the complex between ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 N epsilon H+ activates O2'-H, (d) Glu58 carboxylate acts as base and His92 N epsilon H+ as acid in a general acid-base catalysis. The crystals have the monoclinic space group P2(1), a = 4.968 nm, b = 4.833 nm, c = 4.048 nm, beta = 90.62 degrees with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R = 15.3% with 11,338 data > or = 1 sigma (Fo) in the resolution range 1.0-0.2 nm; this includes 180 water molecules and two Ca2+. The structure of ribonuclease T1 is as previously observed. 3'GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4'-exo puckering, the ribose phosphate is extended with torsion angle epsilon in trans. The O2'-H group is activated by accepting and donating hydrogen bonds from His40 N epsilon H+ and to Glu58 O epsilon 1; the phosphate is hydrogen bonded to Glu58 O epsilon 2H, Arg77 N epsilon H+ and N eta 2H+, Tyr38 O eta H, His92 N eta H+. The conformation of ribose phosphate is such that O2' is at a distance of 0.31 nm from phosphorus, and opposite the P-OP3 bond which accepts a hydrogen bond from His92 N epsilon H+; we infer from a model building study that this bond is equivalent to the scissile P-O5' in a substrate GpN.
核糖核酸酶T1与3'-鸟苷酸（3'GMP）复合物的晶体结构表明：（a）底物GpN以一种积极支持催化过程的构象结合到核糖核酸酶T1的活性位点；（b）反应以线性方式进行；（c）组氨酸40（His40）的NεH⁺激活2'-OH；（d）在一般酸碱催化中，谷氨酸58（Glu58）的羧酸盐作为碱，组氨酸92（His92）的NεH⁺作为酸。晶体属于单斜空间群P2(1)，a = 4.968 nm，b = 4.833 nm，c = 4.048 nm，β = 90.62°，不对称单元中有两个分子。结构通过分子置换法确定，在1.0 - 0.2 nm分辨率范围内，对11338个大于或等于1σ（Fo）的数据进行精修后，R值为15.3%；这包括180个水分子和两个Ca²⁺。核糖核酸酶T1的结构与之前观察到的一样。3'GMP以顺式构象结合；鸟嘌呤位于特定识别位点，核糖采取C4'-外向折叠，核糖磷酸以反式扭转角ε延伸。2'-OH基团通过接受来自His40 NεH⁺的氢键并向Glu58 Oε1供氢而被激活；磷酸与Glu58 Oε2H、精氨酸77（Arg77）的NεH⁺和Nη2H⁺、酪氨酸38（Tyr38）的OηH、His92的NηH⁺形成氢键。核糖磷酸的构象使得2'-O与磷的距离为0.31 nm，且与接受来自His92 NεH⁺氢键的P - OP3键相对；我们从模型构建研究推断，该键等同于底物GpN中的可裂解P - O5'键。
