Publications

• Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.

  Zhang S., Zhou L., Nemeria N., Yan Y., Zhang Z., Zou Y., Jordan F.

  The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) ... >> More

  The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes. << Less

  Biochemistry 44:2237-2243(2005) [PubMed] [EuropePMC]

• Conversion of glyoxylate to hydroxypyruvate by extracts of Escherichia coli.

  BARKULIS S.S., KRAKOW G.

  Biochim Biophys Acta 21:593-594(1956) [PubMed] [EuropePMC]

• C2-alpha-lactylthiamin diphosphate is an intermediate on the pathway of thiamin diphosphate-dependent pyruvate decarboxylation. Evidence on enzymes and models.

  Zhang S., Liu M., Yan Y., Zhang Z., Jordan F.

  Thiamin diphosphate (ThDP)-dependent decarboxylations are usually assumed to proceed by a series of covalent intermediates, the first one being the C2-trimethylthiazolium adduct with pyruvate, C2-alpha-lactylthiamin diphosphate (LThDP). Herein is addressed whether such an intermediate is kinetical ... >> More

  Thiamin diphosphate (ThDP)-dependent decarboxylations are usually assumed to proceed by a series of covalent intermediates, the first one being the C2-trimethylthiazolium adduct with pyruvate, C2-alpha-lactylthiamin diphosphate (LThDP). Herein is addressed whether such an intermediate is kinetically competent with the enzymatic turnover numbers. In model studies it is shown that the first-order rate constant for decarboxylation can indeed exceed 50 s(-1) in tetrahydrofuran as solvent, approximately 10(3) times faster than achieved in previous model systems. When racemic LThDP was exposed to the E91D yeast pyruvate decarboxylase variant, or to the E1 subunit of the pyruvate dehydrogenase complex (PDHc-E1) from Escherichia coli, it was partitioned between reversion to pyruvate and decarboxylation. Under steady-state conditions, the rate of these reactions is severely limited by the release of ThDP from the enzyme. Under pre-steady-state conditions, the rate constant for decarboxylation on exposure of LThDP to the E1 subunit of the pyruvate dehydrogenase complex was 0.4 s(-1), still more than a 100-fold slower than the turnover number. Because these experiments include binding, decarboxylation, and oxidation (for detection purposes), this is a lower limit on the rate constant for decarboxylation. The reasons for this slow reaction most likely include a slow conformational change of the free LThDP to the V conformation enforced by the enzyme. Between the results from model studies and those from the two enzymes, it is proposed that LThDP is indeed on the decarboxylation pathway of the two enzymes studied, and once LThDP is bound the protein needs to provide little assistance other than a low polarity environment. << Less

  J Biol Chem 279:54312-54318(2004) [PubMed] [EuropePMC]

• GLYOXYLATE CARBOLIGASE OF ESCHERICHIA COLI: A FLAVOPROTEIN.

  GUPTA N.K., VENNESLAND B.

  J Biol Chem 239:3787-3789(1964) [PubMed] [EuropePMC]

• Glyoxylate carboligase lacks the canonical active site glutamate of thiamine-dependent enzymes.

  Kaplun A., Binshtein E., Vyazmensky M., Steinmetz A., Barak Z., Chipman D.M., Tittmann K., Shaanan B.

  Thiamine diphosphate (ThDP), a derivative of vitamin B1, is an enzymatic cofactor whose special chemical properties allow it to play critical mechanistic roles in a number of essential metabolic enzymes. It has been assumed that all ThDP-dependent enzymes exploit a polar interaction between a stri ... >> More

  Thiamine diphosphate (ThDP), a derivative of vitamin B1, is an enzymatic cofactor whose special chemical properties allow it to play critical mechanistic roles in a number of essential metabolic enzymes. It has been assumed that all ThDP-dependent enzymes exploit a polar interaction between a strictly conserved glutamate and the N1' of the ThDP moiety. The crystal structure of glyoxylate carboligase challenges this paradigm by revealing that valine replaces the conserved glutamate. Through kinetic, spectroscopic and site-directed mutagenesis studies, we show that although this extreme change lowers the rate of the initial step of the enzymatic reaction, it ensures efficient progress through subsequent steps. Glyoxylate carboligase thus provides a unique illustration of the fine tuning between catalytic stages imposed during evolution on enzymes catalyzing multistep processes. << Less

  Nat Chem Biol 4:113-118(2008) [PubMed] [EuropePMC]

• Reaction mechanisms of thiamin diphosphate enzymes: new insights into the role of a conserved glutamate residue.

  Shaanan B., Chipman D.M.

  Subsequent to the demonstration in the late 1950s of the catalytic power of the C2 anion/ylid of thiamin diphosphate, further convincing evidence was provided demonstrating that the 4'-aminopyrimidine group plays a vital role in activation of this cofactor. Structural evidence from several crystal ... >> More

  Subsequent to the demonstration in the late 1950s of the catalytic power of the C2 anion/ylid of thiamin diphosphate, further convincing evidence was provided demonstrating that the 4'-aminopyrimidine group plays a vital role in activation of this cofactor. Structural evidence from several crystal structures of thiamin diphosphate-dependent enzymes emphasized the presence of a glutamate residue in hydrogen-bonding distance from N1' as a conserved element in these enzymes. The important role of this conserved glutamate in promoting C2-H ionization and activation of thiamin diphosphate was emphasized by site-directed mutagenesis studies. This role was further elaborated by spectroscopic studies of the 4'-aminopyrimidine-iminopyrimidine tautomerization. The low polarity of the environment of the protein-bound thiazolium is an additional factor in the stabilization of the C2 anion/ylid. The recently determined crystal structure and mutagenesis studies of glyoxylate carboligase, in which the position of the conserved glutamate is occupied by valine, now show that, for the multi-step reaction catalyzed by this enzyme, the advantages of accelerating the ionization of C2-H by re-introducing a carboxylate are outweighed by the apparent overstabilization of intermediates. << Less

  FEBS J 276:2447-2453(2009) [PubMed] [EuropePMC]