Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate–thiamin diphosphate adduct in AHAS I

Abstract

Acetohydroxy acid synthase (AHAS; http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/2/1/6.html) is a thiamin diphosphate (ThDP)‐dependent decarboxylase‐ligase that catalyzes the first common step in the biosynthesis of branched‐chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl‐ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2‐ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2‐ketobutyrate (2‐KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing–quench NMR method or stopped‐flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2‐lactyl‐ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2‐KB, putting into question whether AHAS operates via a pure ping–pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor–acceptor:enzyme complex followed by covalent, ThDP‐promoted catalysis with concerted decarboxylation–carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS.

High Resolution Crystal Structures of the Acetohydroxyacid Synthase‐Pyruvate Complex Provide New Insights into Its Catalytic Mechanism

“…These interpretations are fully consistent with a mechanism of catalysis involving allosteric communication between subunits with the functioning enzyme alternating between two asymmetric conformations ,. In this context, a crucial assumption from the asymmetry of FAD observed in CC_1 and CC_2 (Figure ) is that FAD alternates between two different conformations during catalysis.…”

“…In the second stage, the acceptor substrate, pyruvate or 2‐ketobutyrate, reacts with HE‐ThDP to produce 2‐acetolactate or 2‐acetohydroxybutyrate, respectively (Figure ) . Belenky, Steinmetz, Vyazmensky, Barak, Tittmann, Chipman, have proposed that rather than AHAS operating via a pure ping‐pong mechanism, a ternary donor‐acceptor:enzyme complex is formed allowing the decarboxylation (first stage) and carboligation (second stage) to be concerted. Another conclusion arising from their studies is the possibility of an allosteric interaction occurring between the catalytic sites in AHAS.…”

Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity

“…It is notable that for both variants decarboxylation of MThDP (k3′) is significantly faster than other steps in the reaction (Table 2). Further, even though the rate for T377L/A460Y has decreased considerably compared to that of the wild-type enzyme it is still faster than decarboxylation of LThDP by pyruvate-utilizing enzymes [20,31,32]. Intermediates and cofactor were isolated from the protein by acid quench after a reaction time of 1 s and analyzed by 1 H NMR at pH 0.75 at 25 °C as described in Materials and Methods.…”

“…These results are in sharp contrast with those found in previous studies with ZmPDC [24] and ScPDC [33] where formation of LThDP was the fast step and both decarboxylation and product release were appreciably slower ( Table 2). That said, it is not without precedent, as initial C-C bond formation (k 2 ) is the slow step in the reaction of acetohydroxyacid synthase (AHAS) with pyruvate [32]. Finally, it is notable that decarboxylation of LThDP catalyzed by BFDC T377L/A460Y is the fast step in the overall process, and only approximately four-fold slower when compared to ScPDC.…”

Artificial concurrent catalytic processes involving enzymes

“…However, up to 50% yield of 20 (1.5 mM substrate concentration) was obtained with the other approaches. 46,47 Under conditions which included only 10 mM pyruvate but 20 mM benzaldehyde and 50 mM D-alanine a product titre of 14 mM at 5.5 mM benzylamine could be reached. These three enzymes were coexpressed in E. coli and resting cells employed in catalysis.…”