Computational determination of fundamental pathway and activation barriers for acetohydroxyacid synthasecatalyzed condensation reactions of α-keto acids

Acetohydroxyacid synthase (AHAS) is the first common enzyme in the biosynthetic pathway leading to the production of various branched-chain amino acids. AHAS is recognized as a promising target for new antituberculosis drugs, antibacterial drugs, and herbicides. Extensive first-principles quantum mechanical (QM) and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations have enabled us, in this study, to uncover the fundamental reaction pathway, determine the activation barriers, and obtain valuable insights concerning the specific roles of key amino acid residues for the common steps of AHAS-catalyzed condensation reactions of a-keto acids. The computational results reveal that the rate-determining step of the AHAS-catalyzed. reactions is the second reaction step and that the most important amino acid residues involved in the catalysis include Glu.1.44', Gln207', GIy 121', and GIySl 1 that form favorable hydrogen bonds with the reaction center (consisting of atoms from the substrate and cofactor) during the reaction process. In addition, Glu 144' also accepts a proton from cofactor thiamin diphosphate (ThDP) through hydrogen bonding during the catalytic reaction. The favorable interactions between the reaction center and protein environment remarkably stabilize the transition state and, thus, lower the activation barrier for the rate-determining reaction step by ~20 kcal/mol. The activation barrier calculated, for the rate-determining step is in good agreement with the experimental activation barrier. The detailed structural and mechanistic insights should be valuable for rational design of novel, potent AHAS inhibitors that may be used as promising new antituberculosis drugs, antibacterial drugs, and/or herbicides to overcome drug resistance problem.