Exploitation of binding energy for catalysis and design

Thyme, Summer B., et all. Nature 461, 1300-4. (2009)

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*. 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*. The I-AniI enzyme (pdb 2QOJ) is shown here with its substrate. The target site is colored based on the effect of single base-pair substitutions to kcat*, normalized by the sum of the effects on kcat* and KM* ([|Δln(kcat*)| / |(Δln(KM*)|+ |Δln(kcat*)|)]). Most single base substitutions in the target affect kcat* (blue, (+) side) or KM* (red, (-) side) but not both.