Hancox E L, Halford S E
Department of Biochemistry, University of Bristol, U.K.
Biochemistry. 1997 Jun 17;36(24):7577-85. doi: 10.1021/bi970156k.
The EcoRV endonuclease contacts the minor groove of DNA through a peptide loop encompassing residues 67-72. This loop adapts to distorted DNA in the specific complex and to regular DNA in the nonspecific complex. Random mutagenesis had previously identified glutamine 69 as the key component of the loop and this study reports on mutants with glutamate (Q69E), lysine (Q69K), or leucine (Q69L) at this position. The mutants bound DNA specifically at the EcoRV recognition site in the presence of Ca2+, in the same manner as wild-type EcoRV. In the absence of divalent metals, Q69K and Q69L showed the same nonspecific binding as native EcoRV while Q69E failed to bind DNA. Glutamate at position 69 presumably repels nonspecific DNA whilst allowing the adaptations to specific DNA. Both Q69E and Q69K had severely impaired DNA cleavage activities, while Q69L had a steady-state k(cat) within an order of magnitude of wild-type EcoRV though its primary product was nicked DNA, in contrast to double strand breaks by wild-type EcoRV. The activity of Q69L required higher concentrations of Mg2+ than the wild-type and showed a sigmoidal dependence upon the Mg2+ concentration, indicating two metal ions per strand scission. Transient kinetics on Q69L gave lower rate constants for phosphodiester hydrolysis than wild-type EcoRV and its reaction also involved a slow conformational change preceding DNA cleavage that had no equivalent with the wild-type. Gln69 in EcoRV thus plays key roles in the adjustments of the protein to varied DNA structures and in the alignment of the catalytic functions for DNA cleavage.
EcoRV核酸内切酶通过一个包含67 - 72位残基的肽环与DNA的小沟接触。该环在特异性复合物中适应扭曲的DNA，在非特异性复合物中适应规则的DNA。随机诱变先前已确定谷氨酰胺69是该环的关键成分，本研究报道了该位置具有谷氨酸（Q69E）、赖氨酸（Q69K）或亮氨酸（Q69L）的突变体。在Ca2+存在的情况下，这些突变体以与野生型EcoRV相同的方式特异性结合EcoRV识别位点的DNA。在没有二价金属的情况下，Q69K和Q69L表现出与天然EcoRV相同的非特异性结合，而Q69E未能结合DNA。69位的谷氨酸可能排斥非特异性DNA，同时允许对特异性DNA进行适应。Q69E和Q69K的DNA切割活性均严重受损，而Q69L的稳态k(cat)与野生型EcoRV相差一个数量级，尽管其主要产物是带切口的DNA，这与野生型EcoRV产生的双链断裂不同。Q69L的活性需要比野生型更高浓度的Mg2+，并且对Mg2+浓度呈现出S形依赖性，表明每条链断裂需要两个金属离子。对Q69L的瞬态动力学研究表明，其磷酸二酯水解的速率常数低于野生型EcoRV，并且其反应在DNA切割之前还涉及一个缓慢的构象变化，这在野生型中不存在。因此，EcoRV中的Gln69在蛋白质对不同DNA结构的调整以及DNA切割催化功能的排列中起着关键作用。
