Exploitation of binding energy for catalysis and design

Enzymes utilize substrate binding energy both to promote ground state association and to selectively lower the energy of the reaction transition state.i 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.ii 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 K(D) and K(M)*, and those in the (+) half-site primarily decrease 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 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 K(M)*, while 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.