Jeltsch A, Alves J, Wolfes H, Maass G, Pingoud A
Zentrum Biochemie, Medizinische Hochschule Hannover, Germany.
Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8499-503. doi: 10.1073/pnas.90.18.8499.
The crystal structure analyses of the EcoRI-DNA and EcoRV-DNA complexes do not provide clear suggestions as to which amino acid residues are responsible for the activation of water to carry out the DNA cleavage. Based on molecular modeling, we have proposed recently that the attacking water molecule is activated by the negatively charged pro-Rp phosphoryl oxygen of the phosphate group 3' to the scissile phosphodiester bond. We now present experimental evidence to support this proposal. (i) Oligodeoxynucleotide substrates lacking this phosphate group in one strand are cleaved only in the other strand. (ii) Oligodeoxynucleotide substrates carrying an H-phosphonate substitution at this position in both strands and, therefore, lacking a negatively charged oxygen at this position are cleaved at least four orders of magnitude more slowly than the unmodified substrate. These results are supported by other modification studies: oligodeoxynucleotide substrates with a phosphorothioate substitution at this position in both strands are cleaved only if the negatively charged sulfur is in the RP configuration as shown for EcoRI [Koziolkiewicz, M. & Stec, W.J. (1992) Biochemistry 31, 9460-9466] and EcoRV (B. A. Connolly, personal communication). As the phosphate residue 3' to the scissile phosphodiester bond is not needed for strong DNA binding by both enzymes, these findings strongly suggest that this phosphate group plays an active role during catalysis. This proposal, furthermore, gives a straightforward explanation of why in the EcoRI-DNA and EcoRV-DNA complexes the DNA is distorted differently, but in each case the 3' phosphate group closely approaches the phosphate group that is attacked. Finally, an alternative mechanism for DNA cleavage involving two metal ions is unlikely in the light of our finding that both EcoRI and EcoRV need only one Mg2+ per active site for cleavage.
EcoRI-DNA和EcoRV-DNA复合物的晶体结构分析并未明确指出哪些氨基酸残基负责激活水以进行DNA切割。基于分子模型，我们最近提出，进攻水分子是由位于可切割磷酸二酯键3'端的磷酸基团带负电荷的前-Rp磷酰氧激活的。我们现在提供实验证据来支持这一观点。（i）一条链中缺少该磷酸基团的寡脱氧核苷酸底物仅在另一条链中被切割。（ii）两条链在该位置都带有H-膦酸酯取代、因此在该位置缺少带负电荷氧的寡脱氧核苷酸底物，其切割速度比未修饰的底物至少慢四个数量级。其他修饰研究也支持这些结果：两条链在该位置都带有硫代磷酸酯取代的寡脱氧核苷酸底物，只有当带负电荷的硫处于Rp构型时才会被切割，如EcoRI [Koziolkiewicz, M. & Stec, W.J. (1992) Biochemistry 31, 9460-9466] 和EcoRV（B. A. Connolly，个人交流）所示。由于这两种酶与DNA的强结合并不需要位于可切割磷酸二酯键3'端的磷酸残基，这些发现强烈表明该磷酸基团在催化过程中起积极作用。此外，这一观点直接解释了为什么在EcoRI-DNA和EcoRV-DNA复合物中DNA的扭曲方式不同，但在每种情况下，3'磷酸基团都紧密靠近被攻击的磷酸基团。最后，鉴于我们发现EcoRI和EcoRV每个活性位点切割DNA仅需要一个Mg2+，涉及两个金属离子的DNA切割替代机制不太可能成立。
