Bugreev D V, Nevinsky G A
Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
Biochemistry (Mosc). 1999 Mar;64(3):237-49.
X-Ray structure analysis is one of the most informative methods for investigation of enzymes. However, it does not provide quantitative estimation of the relative efficiency of formation of contacts revealed by this method, and when interpreting the data this does not allow taking into account the relative contribution of some specific and nonspecific interactions to the total affinity of nucleic acids (NA) to enzymes. This often results in unjustified overestimation of the role of specific enzyme--NA contacts in affinity and specificity of enzyme action. In recent years we have developed new approaches to analysis of the mechanisms of protein--nucleic acid interactions allowing quantitative estimation of the relative contribution of virtually every nucleotide unit (including individual structural elements) to the total affinity of enzymes to long DNA and RNA molecules. It is shown that the interaction between enzymes and NA on the molecular level can be successfully analyzed by the methods of synthesis and analysis, that is, step-by-step simplification or complication of the structure of a long NA-ligand. This approach allows the demonstration that complex formation including formation of contacts between enzymes and specific NA units can provide neither high affinity of the enzymes to NA nor the specificity of their action. Using a number of sequence-independent replication and repair enzymes specifically recognizing a modified unit in DNA and also some sequence-dependent topoisomerization and restriction enzymes as examples, it was shown that virtually all nucleotide units within the DNA binding cleft interact with the enzyme, and high affinity mainly (up to 5-7 of 7-10 orders of magnitude) is provided by many weak additive interactions between these enzymes and various structural elements of the individual NA nucleotide units. At the same time, the relative contribution of specific interactions to the total affinity of NA is rather small and does not exceed 1-2 orders of magnitude. Specificity of enzyme action is provided by the stages of the enzyme-dependent NA adaptation to the optimal conformation and directly of catalysis: kcat increases by 3-7 orders of magnitude when changing from nonspecific to specific NA. In the present work we summarized our experience in studies of enzymes by the method of step-by-step complication of the ligand structure and performed a detailed analysis of the features of this approach and its possibilities for the study of protein--nucleic acid interactions on the molecular level.
X射线结构分析是研究酶的最具信息量的方法之一。然而，它无法对该方法所揭示的接触形成的相对效率进行定量评估，并且在解释数据时，这使得无法考虑某些特异性和非特异性相互作用对核酸（NA）与酶的总亲和力的相对贡献。这常常导致对特异性酶-NA接触在酶作用的亲和力和特异性中所起作用的不合理高估。近年来，我们开发了新的方法来分析蛋白质-核酸相互作用的机制，从而能够定量评估几乎每个核苷酸单元（包括单个结构元件）对酶与长DNA和RNA分子的总亲和力的相对贡献。结果表明，通过合成和分析方法，即逐步简化或复杂化长NA配体的结构，可以成功地在分子水平上分析酶与NA之间的相互作用。这种方法能够证明，包括酶与特定NA单元之间形成接触在内的复合物形成，既不能提供酶对NA的高亲和力，也不能提供其作用的特异性。以一些特异性识别DNA中修饰单元的与序列无关的复制和修复酶以及一些与序列有关的拓扑异构酶和限制酶为例，结果表明，DNA结合裂隙内几乎所有核苷酸单元都与酶相互作用，而这些酶与单个NA核苷酸单元的各种结构元件之间的许多弱加性相互作用主要提供了高亲和力（高达7-10个数量级中的5-7个数量级）。同时，特异性相互作用对NA总亲和力的相对贡献相当小，不超过1-2个数量级。酶作用的特异性由依赖于酶的NA适应最佳构象以及直接催化的阶段提供：当从非特异性NA转变为特异性NA时，催化常数（kcat）增加3-7个数量级。在本工作中，我们总结了通过逐步复杂化配体结构来研究酶的经验，并对该方法的特点及其在分子水平上研究蛋白质-核酸相互作用的可能性进行了详细分析。
