Enhanced active-site electric field accelerates enzyme catalysis



Enzymes are extraordinarily proficient and selective catalysts arising from billions of years of evolution. The rational design of proteins that outperform enzymes especially in their native functions remains a grand challenge. The current de novo designed enzymes generally exhibit relatively poor activities. This can be improved by directed evolution; however, this is not grounded in an underlying physical principle. In this work, we exploited the physical principle of electrostatic catalysis, in which the large electric fields exerted by the charged and polar chemical groups in enzyme active sites preferentially stabilize the more charge-separated transition state over the reactant state and thus accelerate reactions. We report enhancements in electric fields in the active site of horse liver alcohol dehydrogenase (LADH), probed using the vibrational Stark effect, by changing two key features: replacing the serine hydrogen bond donor with threonine (S48T) and replacing the catalytic Zn2+ with Co2+. We found that these enhanced electric fields accelerate the rate of hydride transfer, an observation substantially reinforcing the theory of electrostatic catalysis: quantitative predictions can be made beyond the scope of naturally occurring enzymes. The effects of the H-bond and the metal coordination, two distinct chemical forces, can be unified using the same physical quantity — electric field, which is quantitative, and shown here to be additive, and predictive. These results suggest a new design paradigm for both biological and non-biological catalysts.


Supplementary material

Supporting Information
Protein expression, metal replacement, protein characterization, vibrational spectroscopies, and enzyme kinetics