The thiamin diphosphate (ThDP)-dependent biosynthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP-), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP- from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP- is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg276) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound HEThDP- with benzaldehyde to form phenylacetylcarbinol (Engel, S., Vyazmensky, M., Geresh, S., Barak, Z., and Chipman, D. M. (2003) Biotechnol. Bioeng. 84, 833-840) provides a new tool for studying the fate of HEThDP- in AHAS, since the formation of the new product has a very different dependence on active site modifications than does acetohydroxyacid acid formation. The effects of mutagenesis of four different residues in the site on the rates and specificities of the normal and unnatural reactions support a critical role for Arg276 in the stabilization of the transition states for ligation of the incoming second ketoacid with HEThDP- and/or for the breaking of the product-ThDP bond. This information makes it possible to engineer the active site so that it efficiently and preferentially catalyzes a new reaction.