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

Abstract

The thiamin diphosphate (ThDP)-dependent enzyme acetohydroxyacid synthase (AHAS) catalyzes the first common step in branched-chain amino acid biosynthesis. By specific ligation of pyruvate with the alternative acceptor substrates 2-ketobutyrate and pyruvate, AHAS controls the flux through this branch point and determines the relative rates of synthesis of isoleucine, valine, and leucine, respectively. We used detailed NMR analysis to determine microscopic rate constants for elementary steps in the reactions of AHAS II and mutants altered at conserved residues Arg-276, Trp-464, and Met-250. In Arg276Lys, both the condensation of the enzyme-bound hydroxyethyl-ThDP carbanion͞enamine (HEThDP) with the acceptor substrates and acetohydroxyacid release are slowed several orders of magnitude relative to the wild-type enzyme. We propose that the interaction of the guanidinium moiety of Arg-264 with the carboxylate of the acceptor ketoacid provides an optimal alignment of substrate and HEThDP orbitals in the reaction trajectory for acceptor ligation, whereas its interaction with the carboxylate of the covalent HEThDP-acceptor adduct plays a similar role in product release. Both Trp-464 and Met-250 affect the acceptor specificity. The high preference for ketobutyrate in the wild-type enzyme is lost in Trp464Leu as a consequence of similar forward rate constants of carboligation and product release for the alternative acceptors. In Met250Ala, the turnover rate is determined by the condensation of HEThDP with pyruvate and release of the acetolactate product, whereas the parallel steps with 2-ketobutyrate are considerably faster. We speculate that the specificity of carboligation and product liberation may be cumulative if the former is not completely committed.mechanism ͉ thiamin diphosphate ͉ amino acid biosynthesis A cetohydroxyacid (AHA) synthase (AHAS), which catalyzes the first common step in the biosynthesis of branched-chain amino acids, belongs to a homologous family of thiamin diphosphate (ThDP)-dependent enzymes whose initial step is decarboxylation of pyruvate or another 2-ketoacid (1-3). In the process catalyzed by AHAS, the decarboxylation of pyruvate to form the hydroxyethyl-ThDP Ϫ anion͞enamine (HEThDP Ϫ ) is followed by the specific condensation of the intermediate with a second aliphatic ketoacid to form an AHA (Fig. 1). Whereas the role of the enzyme in the first steps in AHAS catalysis, i.e., activation of ThDP, decarboxylation of pyruvate, and formation of HEThDP (3), is comparable with the function of other members of its homologous family (4), the function of the protein in controlling the fate of HEThDP is poorly understood.The competition between the alternative ''second substrates'' 2-ketobutyrate and pyruvate, leading to partition of the flux through AHAS between acetohydroxybutyrate (AHB) and acetolactate (AL), determines the relative rates of formation of isoleucine and of valine, leucine, and pantothenate, respectively ( Fig. 1) (5). Most AHASs show a strong preference for 2-ketobutyrate as the s...

Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase

“…This idea is consistent with the finding that the residues equivalent to R377 and W574 in Escherichia coli AHAS interact with the carboxylate and alkyl side chains, respectively, of the second substrate of AHAS (22). Further, almost all of the residues at herbicide-resistance mutation sites are highly conserved across species, suggesting they may play a role in the normal function of the enzyme.…”

“…However, it should be understood that the selection conditions under which these mutations arise make it probable that mutants with low or no activity will not be isolated. Thus R377 mutations have never been found in herbicide-resistant organisms, because such mutants have little or no activity (22,23).…”

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

“…) are comparable with those of other carboligases that use 2-ketoacid substrates, such as glyoxylate carboligase and isozymes of acetohydroxyacid synthase from various bacteria (17,25,(27)(28)(29)(30). The rates on the unactivated enzyme are similar to those of M. tuberculosis acetohydroxyacid synthase, whereas the activated rates are comparable with those of E. coli enzymes.…”

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

“…This allows us to take advantage of Cleland's theory of net rate constants (35) to isolate the KIE on the net rate constant specifically associated with the radical intermediate. This approach is reminiscent of recent studies regarding the steady-state concentrations of stable intermediates associated with thiaminedependent enzymes (36)(37)(38)(39). Our results suggest that DesII must be in the correct protonation state to form the initial α-hydroxyalkyl radical and that it is subsequently deprotonated either before or concerted with its oxidation (Fig.…”