How Thiamine Diphosphate Is Activated in Enzymes

Kern, Dorothee; Kern, Günther; Neef, H; Tittmann, Kai; Killenberg-Jabs, Margrit; Wikner, Christer; Schneider, Günter; Hübner, Gerhard

doi:10.1126/science.275.5296.67

Cited by 265 publications

(263 citation statements)

References 25 publications

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“…This electronic rearrangement leads to a geometry in the transition state and in the first covalent adduct that is more favorable for hydrogen bond formation between the ␣-hydroxyl oxygen and the 4-NH 2 group. At this stage, the 4-NH 2 group can act in proton transfer to this oxygen atom in a manner similar to that described for the deprotonation of the C2 carbon of the thiazolium ring of ThDP (31). In this way, the negative charge of the oxygen atom at the ␣-carbon is compensated.…”

Section: Discussionmentioning

confidence: 81%

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Fiedler

Thorell

Sandalova

et al. 2002

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

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Section: Discussionmentioning

confidence: 81%

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Fiedler

Thorell

Sandalova

et al. 2002

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

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125

141

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“…Glu411 is essential for catalysis (Kern et al 1997), and is 100% conserved in all enzymes. His26 and His66 are both directly involved in catalysis and the substrate specificity of TK , and are found to be highly conserved in all TK-like enzymes.…”

Section: Alignment and Conservation Within Pp-and Pyr-domainsmentioning

confidence: 99%

Evolutionary Analysis of the TPP-Dependent Enzyme Family

2007

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The evolutionary relationships of the thiamine pyrophosphate (TPP) dependent family of enzymes was investigated by generation of a neighbor joining (NJ) phylogenetic tree using sequences from the conserved pyrophosphate (PP) and pyrimidine (Pyr) binding domains of 17 TPP-dependent enzymes.This represents the most comprehensive analysis of TPP-dependent enzyme evolution to date. The phylogeny was shown to be robust by comparison with maximum likelihood (ML) trees generated for each individual enzyme. The study discerned the order in which enzymes diverged in the progenote population and several instances where enzyme types diverged later, and broadly confirms the evolutionary history proposed recently from structural comparisons alone (Duggleby 2006). The relationship between the PP-and Pyr-domains and the recruitment of additional protein domains was examined using the transketolase C-terminal (TKC) domain as an example. This domain has been recruited by several members of the family and yet forms no part of the active site and has unknown function. Removal of the TKC-domain was found to increase activity towards β-hydroxypyruvate (HPA) and glycolaldehyde (GA). Further truncations of the Pyr-domain yielded several variants with retained activity. This suggests the influence of TKC-domain recruitment on the evolution of the mechanism and specificity of transketolase (TK) has been minor, and that the smallest functioning unit of TK comprises the PP-and Pyr-domains whose evolutionary histories extend to all TPP-dependent enzymes.

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“…It then was mixed with 100 mM pyruvate or 50 mM pyruvate plus 50 mM 2-ketobutyrate in a RFQ 3 chemical quenched-flow machine (Kintek, Austin, TX), and the reaction was stopped by the addition of an equal volume of 12.5% (wt͞vol) trichloroacetic acid͞1 M DCl in D 2 O. Note that after quench under these conditions, the C2-H of ThDP does not exchange with solvent (12). The reaction was quenched at times between 0.1 and 10 sec, to make certain that the intermediate distributions were determined under true steady-state conditions.…”

Section: Methodsmentioning

confidence: 99%

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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How Thiamine Diphosphate Is Activated in Enzymes

Cited by 265 publications

References 25 publications

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Evolutionary Analysis of the TPP-Dependent Enzyme Family

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

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