Exploitation of binding energy for catalysis and design

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Abstract
Enzymes use substrate-binding energy to promote ground-state association and to selectively lower the energy of the reaction transition state. Thyme et al. have determined that for the monomeric homing endonuclease I-AniI, which cleaves double-stranded DNA with high sequence specificity, mutation in the N-terminal domain of the enzyme is responsible for increasing certain kinetic parameters (KD and KM), whereas mutation in the C-terminal domain decreases another kinetic parameter (kcat). This unexpected asymmetry in the utilization of enzyme–substrate binding energy for catalysis may enable researchers to more effectively re-engineer endonucleases to cleave genomic target sites for gene therapy and other biomedical applications. Enzymes use substrate-binding energy to promote ground-state association and to selectively stabilize the reaction transition state. Mutations in the amino-terminal domain of the monomeric homing endonuclease I-AniI, which cleaves with high sequence specificity in the centre of a 20-base-pair DNA target site, are now found to have different effects on the kinetic parameters of the enzyme than those in the carboxy-terminal domain, revealing an unexpected asymmetry in the use of enzyme–substrate binding energy for catalysis. 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.