Wenz C, Selent U, Wende W, Jeltsch A, Wolfes H, Pingoud A
Institut für Biochemie, Justus-Liebig-Universität, Giessen, Germany.
Biochim Biophys Acta. 1994 Sep 13;1219(1):73-80. doi: 10.1016/0167-4781(94)90248-8.
According to the crystal structure analysis of a specific EcoRV/DNA complex, the thymine residues of the recognition sequence -GATATC- are not in direct contact with any amino acid residue of the protein. However, several amino acid residues are sufficiently close that it seemed worthwhile trying to create variants of EcoRV with altered specificity by site-directed mutagenesis. Guided by molecular modelling we have replaced. Asn-188 in the catalytic center of EcoRV by Gln to produce a mutant with a relative preference (compared to wild type EcoRV) for substrates in which one thymine of the recognition sequence is replaced by uracil. We have purified and characterized the resulting N188Q mutant. The selectivity value for the engineered enzyme (the ratio of the kcat/KM values for -GATAUC- versus -GATATC-) differs from that of the wild type enzyme by a factor of more than 200.
根据对特定EcoRV/DNA复合物的晶体结构分析，识别序列-GATATC-中的胸腺嘧啶残基不与该蛋白质的任何氨基酸残基直接接触。然而，有几个氨基酸残基距离足够近，以至于通过定点诱变来创建具有改变特异性的EcoRV变体似乎是值得一试的。在分子建模的指导下，我们进行了替换。将EcoRV催化中心的Asn-188替换为Gln，以产生一种突变体，该突变体相对于野生型EcoRV对识别序列中的一个胸腺嘧啶被尿嘧啶取代的底物具有相对偏好。我们已经纯化并表征了所得的N188Q突变体。工程酶的选择性值（-GATAUC-与-GATATC-的kcat/KM值之比）与野生型酶的选择性值相差超过200倍。
