The regulatory subunit of acetohydroxyacid synthase enhances the catalysis of its catalytic subunit on 2-ketobutyrate

Acetohydroxyacid synthase (AHAS), the rate-limiting enzyme in the biosynthetic pathway of branched-chain amino acids, consists of catalytic subunits (CSU) and regulatory subunits (RSU). In this study, we prepared AHAS holoenzymes of Escherichia coli , Saccharomyces cerevisiae , Bacillus subtilis , and Pseudomonas syringae pv. Tomato DC3000 utilizing different methods. Moreover, we studied the catalytic properties of the four holoenzymes and their corresponding CSUs using pyruvate and 2-KB as a respective substrate. The results showed that the eight enzymes (four holoenzymes plus four CSUs) displayed comparable catalytic activity in pyruvate assays, indicating the presence of the RSUs seems not to influence the catalysis of the proteins obviously; while in 2-ketobutyrate (2-KB) assays, the importance of the RSUs on promoting recognition of the substrate and on luring the reactions into condensation of the substrate via stabilizing the crucial intermediate hydroxypropyl-ThDP has been demonstrated. These observations enhance our understanding of the correlation of the structure and function between AHAS full enzyme and its CSU and RSU. In addition, these findings also supply a theoretical basis for future evolution of the proteins so as to improve their catalytic efficiency and broaden their substrate spectrum.