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 o f branched-chain amino acids in plants but is absent in animals. In this pa per, we examine with theoretical simulation techniques one hypothesis for t he 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 n eighbor. To determine the free-energy profiles for the reaction we used a h ybrid semiempirical quantum mechanical/molecular mechanical (QM/MM) potenti al in conjunction with potential of mean force calculations. To obtain accu rate results we found it necessary to correct the semiempirical QM method w ith a term derived from calculations performed with more precise ab initio quantum chemical methods. For the mechanism we studied, our simulations pre dict that the isomerization occurs simultaneously with a proton transfer to the substrate from a protonated glutamate residue of the protein.