Now, Steven G. Boxer and co-workers systematically enhanced the electric fields in the active site of horse liver alcohol dehydrogenase (LADH) to accelerate the rates of hydride transfer in the reverse reaction transforming aldehydes into alcohols. Hereby, LADH forms a ternary complex with a NADH cofactor and the substrate, whereby the enzyme’s active site Zn+2 and the serine residue at position 48 (S48) interact with the carbonyl functionality (C=O) of the aldehyde. During the reaction, a nucleophilic attack of the NADH hydride on C=O increases the dipole of this functional group in the transition state (μTS) compared to the reactant state (μRS) (pictured). Theoretically, the electric field of the enzyme (Fenz) exerted by Zn2+ and S48 should accelerate the hydride transfer by lowering the free-energy barrier (ΔG‡) according to the equation ΔΔG‡ = −Fenz ⋅ (μTS − μRS). To modify the electric field exerted on the CO bond, the researchers exchanged Zn2+ with other metal ions such as Cd2+ and Co2+. X-ray crystallography confirmed that the protein structures were virtually unaffected by these mutations. By exploiting the vibrational Stark effect and using an inhibitor as the substrate analogue, Steven G. Boxer and co-workers were able to measure the electric field exerted by the enzyme variants on the C=O bond of the substrate. Employing the same approach, the electric field changes caused by single-site mutations of the S48 residue were also monitored. The double mutant LADHCo,S48T revealed that the electric field changes resulting from the single mutations were additive, leading to a 52-fold increase in the hydride transfer rate for acetone hydrogenation compared to the wild-type enzyme.
This study stands out as it not only achieved an improvement of the enzyme´s catalytic rate by electric field engineering, but also experimentally measured the change of this physical property during the process and quantified its effect on catalysis.
This is a preview of subscription content, access via your institution