Abstract
The thiamin diphosphate (ThDP)-dependent bio-synthetic 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 (Arg(276)) 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 Arg(276) 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.
Highlights
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
Despite the similarity of AHASs to, for example, pyruvate decarboxylases and pyruvate oxidases [3,4,5,6], AHASs carry out a specific carboligation reaction in which the decarboxylation of pyruvate is followed by the condensation of the bound hydroxyethyl-ThDP anion/enamine (HEThDPϪ) intermediate with a second aliphatic ketoacid to form an acetohydroxyacid (Fig. 1)
Kinetics of Synthesis of Phenyl Acetyl Carbinol—We have found that, in addition to catalyzing the physiological reactions leading to the formation of acetohydroxybutyrate and acetolactate (Fig. 1) [24], AHAS II can catalyze the formation of Rphenyl acetyl carbinol (R-PAC) from pyruvate and benzaldehyde [18]
Summary
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 role of the enzyme in the first steps in AHAS catalysis (i.e. activation of ThDP [7], decarboxylation of pyruvate, and formation of HEThDPϪ (step 1 in Fig. 1)) is comparable with the function of other members of its homologous family [8], it has been difficult to suggest roles for specific protein residues in the final steps (2 and 3) of the reaction in which the product acetohydroxyacid is formed and released One reason for this uncertainty has been the lack of clear direct information on the structure of the regions of the active site that might be involved in selective reaction of HEThDPϪ with a second ketoacid. For AHAS II, Tittmann et al [17] demonstrated that by far the slowest step is formation of the first covalent intermediate, lactyl-ThDP
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