Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli

Gollop, Natan; Damri, Batsheva; Barak, Ze’ev; Chipman, David M.

doi:10.1021/bi00441a024

Cited by 78 publications

(93 citation statements)

References 27 publications

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“…In other words, the question asked was whether ilv gene expression was as high as it was because arginine (or serine) was already partially limiting. From previous work, it had been known that for E. coli K-12 prototrophic strains, isoleucine was somewhat limiting (27 (20), isozyme III is more effective in forming acetohydroxybutyrate than isozyme I, the only other isozyme present in this strain. Thus, the addition of leucine may accentuate the relative isoleucine deficiency in these strains.…”

Section: Discussionmentioning

confidence: 83%

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

1991

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Three different approaches were used to examine the regulatory effects of the amino acids specified by the peptide-coding region of the leader transcript of the ilvGMEDA operon of Escherichia coli K-12. Gene expression was examined in strains carrying an ilvGMED'-lac operon fusion. In one approach, auxotrophic derivatives were starved of single amino acids for brief periods, and the burst of j-galactosidase synthesis upon adding the missing amino acid was determined. Auxotrophic derivatives were also grown for brief periods with a limited supply of one amino acid (derepression experiments). Finally, prototrophic strains were grown in minimal medium supplemented with single and multiple supplements of the chosen amino acids. Although codons for arginine, serine, and proline are interspersed among the codons for the three branched-chain (regulatory) amino acids, they appeared to have no effect when added in excess to prototrophs or when supplied in restricted amounts to auxotrophs. Deletions removing the terminator stem from the leader removed all ilvspecific control, indicating that the attenuation mechanism is the sole mechanism for ilv-specific control.The specific control of expression of the ilvGMEDA operon in the enteric bacteria is characterized by repression of the operon when the three branched-chain amino acids are in ample supply but derepression when the supply of any one of the three is limited (14,30). This ilv-specific regulation appears to be exclusively a consequence of an attenuation mechanism ( Fig. 1) (4,32,38). In principle, the attenuation mechanism controlling the ilv operon appears to be quite similar to that found to control operons involved in tryptophan (35), leucine (19), phenylalanine (61), histidine (3), and threonine (17) biosynthesis: the leader region specifies a short peptide containing a disproportionate frequency of the amino acids that are found to regulate the operon. Thus, the rate at which the leader can be translated serves to sense whether the regulatory amino acid is in short or ample supply.As emphasized by Landick and Yanofsky (31), an important feature of the attenuation mechanism is the pausing of RNA polymerase at a certain site in the leader region. The pause allows time for the ribosome to initiate translation, and as translation proceeds, the base pairing in the protector (1:2 stem, Fig. 2) is progressively disrupted. Should ribosome movement be retarded, presumably by stalling at a regulatory codon, long enough for the bases in the upstream arm of the terminator (bases 157 to 164) to emerge on the nascent transcript, these bases can pair with those in the downstream arm of the protector (bases 98 to 105) in the antiterminator (2:3 stem, Fig. 3). It is assumed that, in the absence of ribosome stalling (repressing conditions), translation proceeds rapidly enough that the bases in the downstream arm of the protector will also be covered by the ribosome or otherwise be sterically prevented from antiterminator formation and the terminator will be formed instead. Altho...

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Section: Discussionmentioning

confidence: 83%

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

1991

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“…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.…”

Section: [9]mentioning

confidence: 99%

“…It is significant that for AHAS II and at least four other AHASs we have examined, the competition between pyruvate and 2-ketobutyrate does not affect the total rate of formation of AHAs. As the concentration of 2-ketobutyrate is increased in the presence of a fixed pyruvate concentration, AHB replaces AL as the product, but the sum of the rates of formation of the two remains essentially constant (6,8). Such behavior requires that the rate-determining step for enzyme turnover be different from the product-determining step (8).…”

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confidence: 99%

“…As the concentration of 2-ketobutyrate is increased in the presence of a fixed pyruvate concentration, AHB replaces AL as the product, but the sum of the rates of formation of the two remains essentially constant (6,8). Such behavior requires that the rate-determining step for enzyme turnover be different from the product-determining step (8). Unless the rate constants of parallel steps with pyruvate and 2-ketobutyrate as the second substrate are coincidentally identical, it further implies that the rate-determining step occurs before binding of the second substrate.…”

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

Tittmann

Vyazmensky

Hübner

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…2,4 The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate leads to them, R, V AHB /V AL =R ([2-ketobutyrate]/[pyruvate]). Among the three enterobacterial enzymes, only AHAS I has a relatively low R factor of 2.…”

Section: Introductionmentioning

confidence: 99%

Steady-State Kinetics of the Recombinant Acetohydroxy Acid Synthase from Tobacco

2002

Bulletin of the Korean Chemical Society

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Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli

Cited by 78 publications

References 27 publications

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

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

Steady-State Kinetics of the Recombinant Acetohydroxy Acid Synthase from Tobacco

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