REFINED CRYSTAL-STRUCTURE OF THE TRIPHOSPHATE CONFORMATION OF H-RAS P21 AT 1.35 A RESOLUTION - IMPLICATIONS FOR THE MECHANISM OF GTP HYDROLYSIS
REFINED CRYSTAL-STRUCTURE OF THE TRIPHOSPHATE CONFORMATION OF H-RAS P21 AT 1.35 A RESOLUTION - IMPLICATIONS FOR THE MECHANISM OF GTP HYDROLYSIS
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DOI：
10.1002/j.1460-2075.1990.tb07409.x
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发表时间：
1990-08-01
期刊：
影响因子：
11.4
通讯作者：
WITTINGHOFER, A
中科院分区：
文献类型：
--
作者：
PAI, EF;KRENGEL, U;WITTINGHOFER, A
H-ras癌基因蛋白p21与缓慢水解的GTP类似物GppNp复合物的晶体结构在1.35 . ang时确定。决议。211个水分子的电子密度。对于所有在6 . ang之间的数据，该结构已被细化到最终r因子为19.8%。1.35分……核苷酸和镁离子的结合位点被详细地揭示出来。对于氨基酸残基61 ~ 65的拉伸，主链原子的温度因子是平均值16.1 ang的4倍。因为有多种构象。在其中一种构象中，Gln61的侧链与一个水分子接触，水分子正好是攻击γ的亲核试剂。- GTP的磷酸。基于这一观察，我们提出了GTP水解的机制，主要涉及Gln61和gl63作为水在线攻击的激活物质。Thr35、Gly60和Lys16残基上的氢键促进了亲核位移。并提出了GAP提高速率的机制。
The crystal structure of the H-ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 .ANG. resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R-factor of 19.8% for all data between 6 .ANG. and 1.35 .ANG.. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61-65, the temperature factors of backbone atoms are four times the average value of 16.1 .ANG.2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the .gamma.-phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in-line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.