A large increase in enzyme–substrate affinity by protein engineering
A large increase in enzyme–substrate affinity by protein engineering
复制标题
通过蛋白质工程大幅增加酶与底物的亲和力
作者：
A. Wilkinson;A. Fersht;D. Blow;P. Carter;G. Winter
在酪氨酰-tRNA 合成酶中进行了单点突变，使其与其底物 ATP 的亲和力 (KM) 提高了 100 倍。在酪氨酰 tRNA 合成酶（嗜热脂肪芽孢杆菌）的晶体结构中，Thr 51 的侧链羟基似乎与底物中间体酪氨酰腺苷酸的 AMP 部分形成弱氢键。然而，在没有底物的情况下，羟基应与水形成牢固的氢键，这将有利于酶-底物复合物的解离。我们使用寡脱氧核苷酸定向诱变在该位点构建了两个点突变体：一个用于去除羟基（Thr 51 → Ala 51），另一个则扭曲局部多肽主链（Thr 51 → Pro 51）。我们在此报告，两种突变体的活性均有所增加（ATP 的 kcat/KM），但一种突变体 (Pro 51) 显示出 25 倍的大幅增加，这主要是由于 ATP 的 KM 降低。这极大地证明了体外诱变在提高酶对其底物的亲和力方面的潜力。
A single point mutation has been engineered in the tyrosyl-tRNA synthetase that improves its affinity (KM) for its substrate ATP by a factor of 100. In the crystal structure of the tyrosyl tRNA synthetase (of Bacillus stearothermophilus), the side-chain hydroxyl of Thr 51 appears to make a weak hydrogen bond with the AMP moiety of the substrate intermediate, tyrosyl adenylate. In the absence of substrate, however, the hydroxyl group should make a strong hydrogen bond with water which would favour dissociation of the enzyme–substrate complex. We have used oligodeoxynucleotide-directed mutagenesis to construct two point mutants at this site: one to remove the hydroxyl group (Thr 51 → Ala 51) and the other, in addition, to distort the local polypeptide backbone (Thr 51 → Pro 51). We report here that both mutants have increased activity (kcat/KM for ATP) but one mutant (Pro 51) shows a massive 25-fold increase due mainly to a lowered KM for ATP. This demonstrates dramatically the potential of in vitro mutagenesis for improving the affinity of an enzyme for its substrate.