Gromova E S, Kubareva E A, Vinogradova M N, Oretskaya T S, Shabarova Z A
Department of Chemistry, Lomonosov State University, Moscow, USSR.
J Mol Recognit. 1991 Jul-Dec;4(4):133-41. doi: 10.1002/jmr.300040405.
To elucidate the mechanism of action of restriction endonucleases MvaI and EcoRII a study was made of their interaction with a set of synthetic substrates in which the heterocyclic bases or the sugar-phosphate backbone had been modified; individual nucleotide residues had been removed or replaced with hydrocarbon bridges, and mismatched base pairs had been introduced. The groups of atoms in the heterocyclic bases and the phosphates in the recognition site that produce the most significant influence on the functioning of endonucleases MvaI and EcoRII were discerned. Profound differences were found in the functioning of the MvaI and EcoRII neoschizomers. The catalytic activity of EcoRII is significantly affected by any alteration in the recognition site structure and conformation, with a modification in one strand of the substrate causing the same decrease in the hydrolysis rate of both strands. Endonuclease MvaI is tolerant to a number of structural abnormalities; the latter sometimes affect only hydrolysis of one strand of the recognition site. The enzyme can preferentially cleave one of the substrate strands. Mismatched base pairs retard and sometimes block the hydrolysis. The effect depends on the particular enzyme, mismatch and its location.
为阐明限制性内切酶MvaI和EcoRII的作用机制，对它们与一组合成底物的相互作用进行了研究，这些底物中的杂环碱基或糖磷酸骨架已被修饰；单个核苷酸残基已被去除或被烃桥取代，并且引入了错配碱基对。识别位点中对MvaI和EcoRII内切酶功能产生最显著影响的杂环碱基中的原子基团和磷酸基团被识别出来。发现MvaI和EcoRII新裂酶在功能上存在深刻差异。EcoRII的催化活性受到识别位点结构和构象任何改变的显著影响，底物一条链上的修饰会导致两条链的水解速率同等程度降低。内切酶MvaI对多种结构异常具有耐受性；后者有时仅影响识别位点一条链的水解。该酶可优先切割底物的一条链。错配碱基对会延迟甚至有时会阻断水解。其影响取决于特定的酶、错配及其位置。
