Role of a Conserved Arginine in the Mechanism of Acetohydroxyacid Synthase

Abstract

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 (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 Acetohydroxyacid synthase (AHAS) 1 belongs to a homologous family of thiamin diphosphate (ThDP)-dependent enzymes that catalyze reactions whose initial step is decarboxylation of pyruvate or another 2-ketoacid (1, 2). However, despite the similarity of AHASs to, for example, pyruvate decarboxylases and pyruvate oxidases (3-6), 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 (HEThDP Ϫ ) intermediate with a second aliphatic ketoacid to form an acetohydroxyacid (Fig. 1). 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 HEThDP Ϫ (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 HEThDP Ϫ with a second ketoacid. Although we have proposed a homology model for AHAS isozyme II from Escherichia coli (9), based on the crystal structure of pyruvate oxidase from Lactobacillus plantarum (LpPOX) (4), these two proteins have very different sequences in the region that is likely to interact with the second substrate. In the first published crystal structure of an AHAS, that of the catalytic subunits of the yeast enzyme (10), this region was disordered. The recent publication of a new structure of the yeast enzyme with a tightly bound herbicide (11) now provides a solid framework for consideration of the role of the protein in directing the fate of the HEThDP Ϫ intermediate. A second, equally serious obstacle to the understanding of the mechanism of AHAS has been a lack of experimental tools for studying the rates of individual steps in the reaction ...

The carboligation reaction of acetohydroxyacid synthase II: Steady-state intermediate distributions in wild type and mutants by NMR

“…The mutation of Met-250, Arg-276, or Phe-109 has a very different effect. The specific activity for formation of AL with pyruvate as sole substrate is decreased by 10-to 100-fold in some mutants at these positions (11). In mutants Met250Ala and Arg276Lys, k cat ͞K M is reduced some 40-fold, but the apparent specificity for reaction with 2-ketobutyrate is changed slightly or not at all (Table 1).…”

“…The construction of plasmids pRSET-GM and pQEV-GM, both of which express the same protein with a sequence identical to the original AHAS II enzyme of E. coli ilvG2096 fused at its N terminus to the pRSET hexahistidine leader, is described in ref. 11. The preparation of all of the site-directed mutants discussed here is as described in ref.…”

“…Many other mutants of AHAS II, altered at Phe-109, Met-250, or Arg-276, exhibit similar behavior: low activity in the formation of AL and a total rate of AHA formation that increases with *The WT AHAS and the mutated enzymes were expressed and purified either as the natural holoenzyme (4) or as a protein with an N-terminal hexahistidine fusion (HisЈ-) (11). Some of this data has appeared in a previous publication (11).…”

“…Some of this data has appeared in a previous publication (11). † These kinetic parameters were determined in steady-state experiments using the colorimetric assay for acetoin (17), with 100 mM pyruvate as the sole substrate, in the presence of 0.1 M KPi buffer (pH 7.6) containing 10 mM MgCl 2, 0.1 mM ThDP, and 75 M FAD, except for variation of the relevant reactant.…”

“…The parameters have experimental uncertainties of 5-10%. ‡ R is the substrate specificity for 2-ketobutyrate as second substrate, (AHB formed)͞(AL formed) ϭ R ϫ [2-ketobutyrate]͞[pyruvate], and was determined by measuring AHB and AL formation simultaneously (8) in a competition experiment with varying 2-ketobutyrate and 50 mM pyruvate, in 0.1 M KPi buffer (pH 7.6) containing 10 mM MgCl 2, 0.1 mM ThDP, and 75 M FAD (11). § Vb͞Va is the ratio between the rate of AL formation with 50 mM pyruvate as sole substrate and the extrapolated rate of AHB formation in the presence of 50 mM pyruvate at saturation with 2-ketobutyrate.…”