A Hybrid-Potential Free-Energy Study of the Isomerization Step of the Acetohydroxy Acid Isomeroreductase Reaction

The enzyme acetohydroxy acid isomeroreductase is a promising target for the design of herbicides because it is an essential enzyme for the synthesis of branched-chain amino acids in plants but is absent in animals. In this paper, we examine with theoretical simulation techniques one hypothesis for the mechanism of the purported rate-limiting step in the reaction catalyzed by the enzyme namely, the isomerization of the deprotonated substrate which involves the migration of a methyl or ethyl group from one carbon to its neighbor. To determine the free-energy profiles for the reaction we used a hybrid semiempirical quantum mechanical/molecular mechanical (QM/MM) potential in conjunction with potential of mean force calculations. To obtain accurate results we found it necessary to correct the semiempirical QM method with a term derived from calculations performed with more precise ab initio quantum chemical methods. For the mechanism we studied, our simulations predict that the isomerization occurs simultaneously with a proton transfer to the substrate from a protonated glutamate residue of the protein.