Vermote C L, Vipond I B, Halford S E
Department of Biochemistry, University of Bristol, U.K.
Biochemistry. 1992 Jul 7;31(26):6089-97. doi: 10.1021/bi00141a019.
A genetic system was constructed for the mutagenesis of the EcoRV restriction endonuclease and for the overproduction of mutant proteins. The system was used to make two mutants of EcoRV, with Ala in place of either Asn185 or Asn188. In the crystal structure of the EcoRV-DNA complex, both Asn185 and Asn188 contact the DNA within the EcoRV recognition sequence. But neither mutation affected the ability of the protein to bind to DNA. In the absence of metal ion cofactors, the mutants bound DNA with almost the same affinity as that of the wild-type enzyme. In the presence of Mg2+, both mutants retained the ability to cleave DNA specifically at the EcoRV recognition sequence, but their activities were severely depressed relative to that of the wild-type. In contrast, with Mn2+ as the cofactor, the mutant enzymes cleaved the EcoRV recognition site with activities that were close to that of the wild-type. When bound to DNA at the EcoRV recognition site, the mutant proteins bound Mn2+ ions readily, but they had much lower affinities for Mg2+ ions than the wild-type enzyme. This was the reason for their low activities with Mg2+ as the cofactor. The arrangement of the DNA recognition functions, at one location in the EcoRV restriction enzyme, are therefore responsible for organizing the catalytic functions at a separate location in the protein.
构建了一个用于EcoRV限制性内切核酸酶诱变和突变蛋白过量表达的遗传系统。该系统用于制备EcoRV的两个突变体，分别用丙氨酸取代Asn185或Asn188。在EcoRV-DNA复合物的晶体结构中，Asn185和Asn188均在EcoRV识别序列内与DNA接触。但这两种突变均未影响蛋白质与DNA结合的能力。在没有金属离子辅因子的情况下，突变体与DNA结合的亲和力几乎与野生型酶相同。在Mg2+存在的情况下，两种突变体均保留了在EcoRV识别序列处特异性切割DNA的能力，但相对于野生型，它们的活性严重降低。相反，以Mn2+作为辅因子时，突变酶切割EcoRV识别位点的活性与野生型接近。当在EcoRV识别位点与DNA结合时，突变蛋白很容易结合Mn2+离子，但它们对Mg2+离子的亲和力比野生型酶低得多。这就是它们以Mg2+作为辅因子时活性较低的原因。因此，EcoRV限制酶中一个位置的DNA识别功能的排列负责在蛋白质的另一个位置组织催化功能。
