Poland B W, Hou Z, Bruns C, Fromm H J, Honzatko R B
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.
J Biol Chem. 1996 Jun 28;271(26):15407-13. doi: 10.1074/jbc.271.26.15407.
Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A. Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base. The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group. Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site. The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites. The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate. No electron density, however, for the corresponding diphosphate nucleoside is observed. In addition, electron density for bound Mg2+ is absent in these nucleotide complexes. The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase. The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase.
已将大肠杆菌腺苷酸琥珀酸合成酶与鸟苷 - 5'-（β，γ - 亚氨基）三磷酸和鸟苷 - 5'-（β，γ - 亚甲基）三磷酸在有和没有Mg2+存在下形成的复合物结构，针对名义分辨率为2.7 Å的数据精修至R因子低于0.2。合成酶的Asp333与鸟嘌呤核苷酸碱基的环外2 - 氨基和环内N1基团形成氢键，而Ser414的羟基和Lys331的主链酰胺与6 - 氧代位置形成氢键。Lys331和Pro417的侧链堆积在鸟嘌呤核苷酸碱基的相对面上。合成酶既不识别鸟嘌呤核苷酸的N7位置，也不识别核糖基团。鸟嘌呤 - 5'-（β，γ - 亚氨基）三磷酸复合物的电子密度与结合到活性位点的三磷酸核苷及其水解的二磷酸核苷的混合物一致。碱基、核糖和α - 磷酸位置重叠，但β - 磷酸占据不同的结合位点。鸟苷 - 5'-（β，γ - 亚甲基）三磷酸与活性位点的结合与鸟苷 - 5'-（β，γ - 亚氨基）三磷酸的结合相当。然而，未观察到相应二磷酸核苷的电子密度。此外，在这些核苷酸复合物中不存在结合Mg2+的电子密度。将合成酶的鸟嘌呤核苷酸复合物与其他GTP结合蛋白的复合物以及GDP、IMP、Mg2+和琥珀酸与合成酶形成的复合物的初步结构进行了比较。在此报道的条件下，该酶不会因鸟嘌呤核苷酸的结合而发生构象变化，并且必须至少存在IMP和/或Mg2+才能促进合成酶对鸟嘌呤核苷酸的完全识别。
