酶利用基质结合能来促进基态结合和选择性地降低反应过渡态的能量。Thyme等人已确定，对单体“归巢”核酸内切酶I-AniI（该酶能以高序列特异性分解双链DNA）来说，这种酶的N-端区域的突变造成某些运动参数（KA 和 KM）增加，而C-端区域的突变则减小另一运动参数（kcat）。在利用酶基质结合能进行催化方面的这一出乎意料的非对称性，也许使研究人员能够更有效地重新设计核酸内切酶，来分解基因组目标点，用于基因疗法和其他生物医学应用。（生物谷Bioon.com）
生物谷推荐原始出处：
Nature 461, 1300-1304 (29 October 2009) | doi:10.1038/nature08508
Exploitation of binding energy for catalysis and design
Summer B. Thyme1,3, Jordan Jarjour2,5, Ryo Takeuchi6, James J. Havranek7, Justin Ashworth1,3, Andrew M. Scharenberg2,5, Barry L. Stoddard3,6 & David Baker1,3,4
1 Department of Biochemistry,
2 Department of Immunology,
3 Graduate Program in Biomolecular Structure and Design,
4 Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA
5 Seattle Children's Hospital Research Institute, 1900 9th Ave M/S C9S-7, Seattle, Washington 98177, USA
6 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, Washington 98109, USA
7 Department of Genetics, Campus Box 8232, Washington University School of Medicine, 4566 Scott Avenue, St Louis, Missouri 63110, USA
Correspondence to: Summer B. Thyme1,3David Baker1,3,4 Correspondence and requests for materials should be addressed to S.B.T. or D.B.
Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively1. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the centre of a 20-base-pair (bp) DNA target site, with the amino (N)-terminal domain of the enzyme making extensive binding interactions with the left (-) side of the target site and the similarly structured carboxy (C)-terminal domain interacting with the right (+) side2. Here we show that, despite the approximate twofold symmetry of the enzyme–DNA complex, 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. Although single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (-) DNA half-site almost exclusively increase the dissociation constant (K D) and the Michaelis constant under single-turnover conditions (K M*), and those in the (+) half-site primarily decrease the turnover number (k cat*). 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 use 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 K M*, whereas redesigns with altered specificities on the (+) side modulate k cat*. Our results illustrate how classical enzymology and modern protein design can each inform the other.
