Pai E F, Krengel U, Petsko G A, Goody R S, Kabsch W, Wittinghofer A
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, FRG.
EMBO J. 1990 Aug;9(8):2351-9. doi: 10.1002/j.1460-2075.1990.tb07409.x.
The crystal structure of the H-ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A 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 A and 1.35 A. 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 A2 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.
已在1.35埃分辨率下确定了与缓慢水解的GTP类似物GppNp复合的H-ras癌基因蛋白p21的晶体结构。已将211个水分子构建到电子密度图中。对于6埃至1.35埃之间的所有数据，该结构已精修至最终R因子为19.8%。核苷酸和镁离子的结合位点得到了高度详细的揭示。对于氨基酸残基61 - 65段，由于多种构象，主链原子的温度因子是16.1埃²平均值的四倍。在这些构象之一中，Gln61的侧链与一个水分子接触，该水分子的位置恰好适合作为亲核试剂攻击GTP的γ-磷酸基团。基于这一观察结果，我们提出了一种GTP水解机制，主要涉及Gln61和Glu63作为水进行线性攻击的活化物种。来自Thr35、Gly60和Lys16残基的氢键促进了亲核取代。还提出了一种由GAP提高反应速率的机制。
