Malygin E G, Ovechkina L G, Evdokimov A A, Zinov'ev V V
Mol Biol (Mosk). 2001 Jan-Feb;35(1):65-78.
Interaction of T4 DNA-(N6-adenine)-methyltransferase [EC 2.1.1] was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k1 = 0.21 s-1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k2 = 0.023 s-1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI Mtase. Substitutions of bases and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme-substrate complexes is usually sharply reduced. The flipping of the adenosine residue, a target for the modification in the recognition site, revealed by fluorescence titration, upon interaction with the enzyme supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.
利用多种含有天然识别位点GATC或其修饰变体的合成寡核苷酸底物，研究了T4 DNA -（N6 -腺嘌呤）-甲基转移酶[EC 2.1.1]的相互作用。在反应过程的十分之一秒和一秒时间间隔内获得的数据，首次使得能够将底物甲基化速率与稳态反应参数进行比较。已确定底物反应分两个阶段进行。由于已表明在稳态条件下T4甲基转移酶形成二聚体结构，因此假定了以下事件序列。当T4甲基转移酶单体与寡核苷酸双链体碰撞时，形成不对称复合物，其中相对于特异性识别位点的一条链随机取向的酶催化甲基从S -腺苷甲硫氨酸快速转移至腺苷残基（k1 = 0.21 s-1）。同时，第二个T4甲基转移酶亚基添加到复合物中，使反应得以继续。在第二阶段过程中，其速度慢一个数量级（对于具有天然位点的双链体，k2 = 0.023 s-1），二聚体T4甲基转移酶切换至第二条链并对第二个残基（靶标）进行甲基化。尽管这种差异远小于EcoRI甲基转移酶所显示的差异，但甲基从供体S -腺苷甲硫氨酸转移至DNA的速率远高于T4甲基转移酶催化的稳态反应的总体速率。识别位点中的碱基取代和缺失以不同方式影响底物参数。当GAT序列被破坏时，初始有生产性的酶 - 底物复合物的比例通常会急剧降低。通过荧光滴定揭示的识别位点中作为修饰靶标的腺苷残基与酶相互作用时的翻转，支持了关于这种DNA变形参与各种DNA -甲基转移酶催化反应的现有观点。
