MAGNESIUM-ION CATALYZED P-N BOND HYDROLYSIS IN IMIDAZOLIDE-ACTIVATED NUCLEOTIDES - RELEVANCE TO TEMPLATE-DIRECTED SYNTHESIS OF POLYNUCLEOTIDES
MAGNESIUM-ION CATALYZED P-N BOND HYDROLYSIS IN IMIDAZOLIDE-ACTIVATED NUCLEOTIDES - RELEVANCE TO TEMPLATE-DIRECTED SYNTHESIS OF POLYNUCLEOTIDES
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DOI：
10.1021/ja00200a053
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发表时间：
1989-08-30
影响因子：
15
通讯作者：
ALBERAS, DJ
中科院分区：
文献类型：
--
作者：
KANAVARIOTI, A;BERNASCONI, CF;ALBERAS, DJ
镁是酶促和非酶促模板导向的多核苷酸合成反应中必需的离子，已被发现可以催化氢氧化物离子攻击核苷酸衍生物的P-N键，如鸟苷5“-单磷酸2-甲基咪唑烷（2-MeImpG），鸟苷5”-单磷酸咪唑烷（ImpG）和腺苷5“-单磷酸2-甲基咪唑烷（2-MeImpA）。钙离子的作用与之相似，但在数量上影响较小。得到了2-MeImpG和ImpG水解的准一级速率常数随Mg2+浓度的变化规律。.ltoreq pH值。摄氏37度，摄氏10度。Mg2+的催化作用在pH值为10时特别有效，其中0.02 M的浓度导致15倍的加速，0.2 M的浓度导致115倍的加速。在其他pH值下，Mg2+的催化作用不那么明显，主要是因为非催化反应更快。Mg2+的催化作用归因于底物的两性离子形式（SH.+-）的反应。（S-，咪唑烷部分质子化）与OH-而不是阴离子形式（S-，咪唑烷部分去质子化）与水反应。这一结论是基于对n -甲基咪唑平衡反应原位生成的N-MeImpG和1,2-二甲基咪唑平衡生成的n -甲基化底物N-MeImpG和1,2-二甲基咪唑平衡生成的2-MeImpG的研究。相反，在没有Mg2+的情况下，S-与水的反应以SH +-的反应完成。哦,。本文研究了Mg2+催化模板定向合成2-MeImpG寡核苷酸和多核苷酸的机理，以及寡核苷酸合成和2-MeImpG水解之间的竞争。
Magnesium, an ion necessary in enzymatic as well as in nonenzymatic template-directed polynucleotide-synthesizing reactions, has been found to catalyze the hydroxide ion attack on the P-N bond of selected 5''-monophosphate imidazolide derivatives of nucleotides, such as guanosine 5''-monophosphate 2-methylimidazolide (2-MeImpG), guanosine 5''-monophosphate imidazolide (ImpG), and adenosine 5''-monophosphate 2-methylimidazolide (2-MeImpA). Calcium ion behaves similarly, but quantitatively the effects are smaller. Pseudo-first-order rate constants of 2-MeImpG and ImpG hydrolysis as a function of Mg2+ concentration have been obtained in the range 6 .ltoreq. pH .ltoreq. 10 at 37.degree.C. Mg2+ catalysis is particularly effective around pH 10 where a 0.02 M concentration leads to 15-fold acceleration and a 0.2 M concentration to a 115-fold acceleration of the rate. At other pH values Mg2+ catalysis is less dramatic, mainly because the noncatalyzed reactions is faster. Mg2+ catalysis is attributed to the reaction of the zwitterionic form of the substrate (SH.+-., imidazolide moiety protonated) with OH- rather than reaction of the anionic form (S-, imidazolide moiety deprotonated) with water. This conclusion is based on a study of the N-methylated substrates N-MeImpG and 1,2-diMeImpG, respectively, which were generated in situ by the equilibrium reaction of ImpG with N-methylimidazole and 2-MeImpG with 1,2-dimethylimidazole, respectively. In contrast, in the absence of Mg2+ the reaction of S- with water completes with the reaction of SH.+-. with OH-. The present study bears on the mechanism of the Mg2+-catalyzed template-directed synthesis of oligo- and polynucleotides derived from 2-MeImpG and on the competition between oligonucleotide synthesis and hydrolysis of 2-MeImpG.