Jeltsch A, Pleckaityte M, Selent U, Wolfes H, Siksnys V, Pingoud A
Institut für Biochemie, Justus-Liebig Universität, Giessen, Germany.
Gene. 1995 May 19;157(1-2):157-62. doi: 10.1016/0378-1119(94)00617-2.
Substrate-assisted catalysis was suggested to be involved in the DNA cleavage reaction of the restriction endonucleases (ENases) EcoRI and EcoRV, because experimental evidence exists that the phosphate group 3' to the scissile bond serves to deprotonate the attacking water. Here, we have addressed the question whether this is a general mechanistic feature of the reactions catalyzed by ENases. For this purpose, the cleavage rates of modified and unmodified oligodeoxyribonucleotides (oligos), in which the phosphate group 3' to the scissile bond is substituted by a methyl phosphonate, were measured for 17 enzymes. Only five turned out not to be inhibited by this modification (BglII, BstI, BstYI, Cfr10I and MunI); all others cleave the modified substrate at a strongly reduced rate or not at all. By employing a hemisubstituted oligo substrate we were able to further investigate the mechanism of inhibition of the latter group of ENases. Some of them cleave the unmodified strand of the modified substrate with a nearly unaltered rate, whereas the modified strand is cleaved very slowly or not at all (BamHI, Bsp143I, Eco72I, MflI, NdeII, Sau3AI, XhoII). The others (AluI, Cfr9I, DpnII, MboI, PvuII) cleave the modified strand of the modified substrate with a largely reduced rate or not at all. These ENases, however, cleave the unmodified strand with a reduced rate, too. Based on these results we conclude that BamHI, Bsp143I, Cfr9I, DpnII, Eco72I, MboI, MflI, NdeII, PvuII, Sau3AI and XhoII may possibly employ substrate assistance in catalysis.
底物辅助催化被认为参与了限制性内切核酸酶（ENases）EcoRI和EcoRV的DNA切割反应，因为有实验证据表明，可切割键3'端的磷酸基团有助于使进攻的水去质子化。在此，我们探讨了这是否是ENases催化反应的普遍机制特征。为此，我们测量了17种酶对修饰和未修饰的寡脱氧核糖核苷酸（寡核苷酸）的切割速率，其中可切割键3'端的磷酸基团被甲基膦酸酯取代。结果发现只有五种酶不受这种修饰的抑制（BglII、BstI、BstYI、Cfr10I和MunI）；其他所有酶切割修饰后的底物的速率都大幅降低或根本不切割。通过使用半取代的寡核苷酸底物，我们能够进一步研究后一组ENases的抑制机制。其中一些酶切割修饰后底物的未修饰链的速率几乎不变，而修饰链的切割非常缓慢或根本不切割（BamHI、Bsp143I、Eco72I、MflI、NdeII、Sau3AI、XhoII）。其他酶（AluI、Cfr9I、DpnII、MboI、PvuII）切割修饰后底物的修饰链的速率大幅降低或根本不切割。然而，这些ENases切割未修饰链的速率也降低。基于这些结果，我们得出结论，BamHI、Bsp143I、Cfr9I、DpnII、Eco72I、MboI、MflI、NdeII、PvuII、Sau3AI和XhoII可能在催化过程中利用了底物辅助。
