Exploitation of binding energy for catalysis and design.
Exploitation of binding energy for catalysis and design.
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作者：
酶利用底物结合能促进基态结合，并选择性地降低反应过渡态的能量。单体归甲内切酶I-AniI在20个碱基对DNA靶位的中心具有高序列特异性，酶的n端结构域与靶位的左（−）侧进行广泛的结合相互作用，类似结构的c端结构域与右（+）侧相互作用。尽管酶- dna复合物具有近似的双重对称性，但我们发现，负责底物与界面（−）侧结合的相互作用和负责过渡态稳定到（+）侧的相互作用几乎完全分离。虽然整个DNA靶位点的单碱基对替换降低了催化效率，但（−）DNA半位点的突变几乎完全增加了KD和KM*，（+）半位点的突变主要降低了kcat*。通过将底物与酵母表面显示的核酸内切酶系在一起，可以抑制（−）侧突变而不是（+）侧突变所产生的活性降低。这种利用酶-底物结合能进行催化的戏剧性不对称与重新设计内切酶以切割基因治疗和其他应用的基因组靶点直接相关。通过计算重新设计的酶通过调节KM*来实现（−）侧的新特异性，而重新设计的酶通过改变（+）侧的特异性来调节kcat*。我们的研究结果说明了经典酶学和现代蛋白质设计是如何相互联系的。
Enzymes utilize substrate binding energy both to promote ground state association and to selectively lower the energy of the reaction transition state. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the center of a 20 base-pair DNA target site, with the N-terminal domain of the enzyme making extensive binding interactions with the left (−) side of the target site and the similarly structured C-terminal domain interacting with the right (+) side. Despite the approximate two-fold symmetry of the enzyme-DNA complex, we find that there is almost complete segregation of interactions responsible for substrate binding to the (−) side of the interface and interactions responsible for transition state stabilization to the (+) side. While single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (−) DNA half-site almost exclusively increase KD and KM*, and those in the (+) half-site primarily decrease kcat*. The reduction of activity produced by mutations on the (−) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the utilization of enzyme-substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (−) side do so by modulating KM*, while redesigns with altered specificities on the (+) side modulate kcat*. Our results illustrate how classical enzymology and modern protein design can each inform the other.
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