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Exploitation of binding energy for catalysis and design


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 (KD) and the Michaelis constant under single-turnover conditions (KM*), and those in the (+) half-site primarily decrease the turnover number (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 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 KM*, whereas 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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Figure 1: Segregation of contributions to binding and catalysis.
Figure 2: Contributions to catalysis.
Figure 3: Computational redesign of specificity.

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This work was supported by a National Science Foundation graduate research fellowship to S.B.T., the US National Institutes of Health (GM084433 and RL1CA133832), the Foundation for the National Institutes of Health through the Gates Foundation Grand Challenges in Global Health Initiative, and the Howard Hughes Medical Institute. We thank A. Quadri for help with plasmid substrate preparation and M. Scalley-Kim for I-AniI cleavage data collected in the presence of Mn2+.

Author Contributions S.B.T and J.J.H. performed computational design calculations and S.B.T. performed kinetic characterization of all designed and wild-type enzymes. R.T. performed the fluorescence competition binding experiment. J.J. performed the surface-expressed tethered cleavage assay. J.A. and J.J.H. developed computational design procedures. S.B.T. and D.B. wrote the paper. Multiple discussions of shared data among all authors at Northwest Genome Engineering Consortium ( group meetings contributed to the recognition of binding/catalysis asymmetry in I-AniI Y2 and the conceptual development of this manuscript.

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Correspondence to Summer B. Thyme or David Baker.

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Thyme, S., Jarjour, J., Takeuchi, R. et al. Exploitation of binding energy for catalysis and design . Nature 461, 1300–1304 (2009).

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