CRYSTALLOGRAPHIC AND BIOCHEMICAL INVESTIGATION OF THE LEAD(II)-CATALYZED HYDROLYSIS OF YEAST PHENYLALANINE TRANSFER-RNA
CRYSTALLOGRAPHIC AND BIOCHEMICAL INVESTIGATION OF THE LEAD(II)-CATALYZED HYDROLYSIS OF YEAST PHENYLALANINE TRANSFER-RNA
复制标题
DOI：
10.1021/bi00339a012
复制
发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
KLUG, A
中科院分区：
文献类型：
--
作者：
BROWN, RS;DEWAN, JC;KLUG, A
收集了在pH5.0和pH7.4的稀醋酸铅（II）溶液中浸泡的酵母tRNAPhe单斜晶体的X射线衍射数据，分辨率为3埃，通过差示傅立叶分析得到了Pb（II）的结合位点。在这两个pH值下观察到相同的三个Pb（II）结合位点。在pH 7.4时，差异图上靠近其中一个Pb（II）结合位点和磷酸-18位置处出现负电子密度的额外峰，表明该衍生物中tRNAPhe分子的残基D-17和G-18之间的糖磷酸链发生断裂。在pH 5.0时，在结构中不会发生任何可观察到的程度的断链，因此，我们得到了该断裂反应的反应物（pH 5.0）和产物（pH 7.4）的图片。聚丙烯酰胺凝胶电泳以及测序实验证实了tRNAPhe分子被切割成四分之一和四分之三的片段，其中较短的片段基本上由残基G-1至D-17组成，而较大的片段包含残基G-18至A-76。端基分析表明，在四分之一片段的D-17处有一个核糖环状2“，3”-磷酸，在四分之三片段的G-18处有一个5“-OH。tRNAPhe分子的裂解不发生在Pb（II）的情况下，这些金属离子之一的裂解位点的接近强烈暗示该金属离子在裂解反应。考虑到上述生物化学和晶体学事实，对反应的几种可能机制的考虑表明，裂解涉及通过Pb（II）结合的羟基从核糖-17的2“-OH去除质子。随后产生的2“-O-对磷酸-18的磷原子进行亲核攻击，可能是通过五配位磷环状中间体（如胰腺核糖核酸酶A的作用），导致断链。它不能决定是否位移，在五坐标中间，通过一个在线或相邻的途径进行，但任何途径的可能性的探索。链断裂在特定的网站偶然发生，因为水铅（II）离子结合在正确的距离，大概是在这样一种方式，以正确的方向提出一个羟基基团，以实现质子提取。Pb（II）结合水分子的pK 1约为7，这将促进在该pH下的反应，但将在pH 5.0下显著减慢反应。的晶体学实验表明，参与裂解反应的Pb（II）离子的占有率减少了约50%的结构在pH值为7.4相比，在pH值为5.0。我们由此得出结论，裂解反应很可能是催化的，而不是化学计量的，因为Pb（II）将在裂解发生后释放，然后可能能够实现进一步的裂解。这一点得到了生物化学实验的证实。这项工作的毒性铅的影响进行了检查。
X-ray diffraction data from monoclinic crystals of yeast tRNAPhe soaked in dilute lead(II) acetate solutions at pH 5.0 and at pH 7.4 have been collected to a resolution of 3 .ANG., and the Pb(II) binding sites have been obtained by difference Fourier analyses. The same three Pb(II) binding sites are observed at both of these pH values. At pH 7.4 an extra peak of negative electron density appears on the difference map close to one of the Pb(II) binding sites and at the position of phosphate-18, indicating cleavage of the sugar-phosphate-chain between residues D-17 and G-18 of the tRNAPhe molecule in this derivative. Chain scission does not occur to any observable extent in the structure at pH 5.0, and we have, therefore, a picture of the reactants (at pH 5.0) and products (at pH 7.4) of this cleavage reaction. Polyacrylamide gel electrophoresis as well as sequencing experiments confirms the cleavage of the tRNAPhe molecule into one-fourth and three-fourth fragments, with the shorter fragment consisting essentially of residues G-1 through D-17 while the larger fragment contains residues G-18 through A-76. End-group analyses suggest a ribose cyclic 2'',3''-phosphate at D-17 of the one-fourth fragment with a 5''-OH at G-18 of the three-fourth fragment. Cleavage of the tRNAPhe molecule does not occur in the absence of Pb(II), and the proximity of one of these metal ions to the cleavage site strongly implicates this metal ion in the cleavage reaction. Consideration of several possible mechanisms for the reaction, taking into account the biochemical and crystallographic facts presented above, suggests that the cleavage involves removal of the proton from the 2''-OH of ribose-17 by a Pb(II)-bound hydroxyl group. Subsequent nucleophilic attack of the resulting 2''-O- on the phosphorus atom of phosphate-18, presumably through a pentacoordinate phosphorus cyclic intermediate (as in the action of pancreatic ribonuclease A), results in chain scission. It cannot be decided whether the displacement, within the pentacoordinate intermediate, proceeds via an in-line or adjacent pathway, but an exploration of the likelihood of either pathway is presented. Strand cleavage at the particular site occurs fortuitously because the aquo Pb(II) ion binds at the correct distance and presumably in such a manner as to present a hydroxyl group in the correct orientation to effect the proton abstraction. The pK1 of a Pb(II)-bound water molecule is approximately 7, which will facilitate reaction at that pH but will slow it substantially at pH 5.0. The crystallographic experiments indicate that the occupancy of the Pb(II) ion involved in the cleavage reaction is reduced by approximately 50% in the structure at pH 7.4 compared with that at pH 5.0. We conclude from this that the cleavage reaction may well be catalytic, rather than stoichiometric, since Pb(II) would be released after cleavage has occurred and may then be able to effect further cleavages. This is corroborated by biochemical experiments. The implications of this work for the toxicity of lead are examined.