A QM/MM study on the last two steps of the catalytic cycle of acetohydroxyacid synthase

The acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that participates in the biosynthetic pathway of branched-chain amino acids. The catalytic cycle of AHAS consists of five steps and the first three steps are analogues to other thiamin diphosphate dependent enzymes, but some mechanism aspect of the last two steps of the AHAS catalytic cycle remain unclear. We have modeled the last two catalytic steps of the AHAS, using a combined quantum mechanical and molecular mechanical method (QM/MM). Substrates (pyruvate and hydroxyethylthiamine diphosphate anion) and three important amino acids (Arg380, Gly116 and Glu139) of the active site were considered into the QM region. A complete characterization of the potential energy surface was performed using the AM1/CHARMM27 approach. In order to improve the semi-empirical calculations, the AM1/MM potential energy surface was corrected at B3LYP/6-31G(d,p) level (B3LYP/6-31G(d,p)//AM1/MM). Our results show that the protein environment is crucial to provide an adequate description of the reaction mechanisms. This fact is consequence of the increase of the substrate polarization by the enzyme environment. In this sense, the largest substrate polarization is observed when selected amino acid residues are included within the QM region in B3LYP/6-31G(d,p)//AM1/MM calculations.