Acetohydroxyacid synthase (AHAS, EC 2.2.1.6 also referred to as acetolactate synthase) catalyzes the first common step in the metabolic pathway leading to biosynthesis of the branched-chain amino acids in plants and microorganisms. Due to its presence in plants, AHAS is a target for the herbicides (sulfonylurea and imidazolinone), which act as potent inhibitors of the enzyme. Recently, we have shown [J. Kim, D.G. Baek, Y.T. Kim, J.D. Choi, M.Y. Yoon, Biochem. J. (2004) 384, 59-68] that the residues in the “mobile loop” 567-582 on the C-termini are involved in the binding/stabilization of the active dimer and ThDP (thiamin diphosphate) binding. In this study, we have demonstrated the role of the W573 in the mobile loop of the C-termini of tobacco AHAS. The substitution of this W573 residue caused significant perturbations in the activation process and in the binding site of ThDP. Position W573 plays a structurally important role in the binding of FAD, maintaining the enzyme active site in the required geometry for catalysis to occur. In here we propose that the tryptophan at position 573 is important for the catalytic process.