Exploitation of binding energy for catalysis and design

摘要: Enzymes use substrate-binding energy both to promote ground-state association and stabilize the reaction transition state selectively. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in centre of a 20-base-pair (bp) DNA target site, amino (N)-terminal domain enzyme making extensive binding interactions left (-) side site similarly structured carboxy (C)-terminal interacting right (+) side. Here we show that, despite approximate twofold symmetry enzyme-DNA complex, there is almost complete segregation responsible for substrate interface transition-state stabilization Although single base-pair substitutions throughout entire reduce catalytic efficiency, mutations half-site exclusively increase dissociation constant (K(D)) Michaelis under single-turnover conditions (K(M)*), those primarily decrease turnover number (k(cat)*). reduction activity produced by on side, but not can be suppressed tethering displayed surface yeast. This dramatic asymmetry enzyme-substrate catalysis has direct relevance redesign endonucleases cleave genomic sites gene therapy other applications. Computationally redesigned enzymes that achieve new specificities do so modulating K(M)*, whereas redesigns altered modulate k(cat)*. Our results illustrate how classical enzymology modern protein design each inform other.