Electronic and nuclear magnetic resonance spectroscopic features of the 1',4'-iminopyrimidine tautomeric form of thiamin diphosphate, a novel intermediate on enzymes requiring this coenzyme

Appropriate compounds were synthesized to create models for the 1',4'-imino tautomer of the 4'-aminopyrimidine ring of thiamin diphosphate recently found to exist on the pathway of enzymatic reactions requiring this cofactor [Jordan, F., and Nemeria, N. S. (2005) Bioorg. Chem. 33, 190-215]. The N1-methyl-4-aminopyrimidinium compounds synthesized on treatment with a strong base produce the 1,4-imino tautomer whose UV spectrum indicates a maximum between 300 and 320 nm, depending on the absence or presence of a methyl group at the 4-amino nitrogen. The lambda(max) found is in the same wavelength range as the positive circular dichroism band observed on several enzymes and showed a very strong dependence on solvent dielectric constant. To help with the 15N chemical shift assignments, the model compounds were specifically labeled with 15N at the amino nitrogen atom. The chemical shift of the amino nitrogen was deshielded by N1-methylation and then dramatically further deshielded by more than 100 ppm on formation of the 1,4-iminopyrimidine tautomer. Both the UV spectroscopic values and the 15N chemical shift for the 1,4-iminopyrimidine tautomer should serve as useful guides to the assignment of enzyme-bound signals.     

Synthesis with good enantiomeric excess of both enantiomers of alpha-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369-7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473-21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and beta-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity

The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K(d,ThDP) = 4.34 microM and K(m,ThDP) = 11 microM were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1',4'-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.     

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

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

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.     

Acetylphosphinate is the most potent mechanism-based substrate-like inhibitor of both the human and Escherichia coli pyruvate dehydrogenase components of the pyruvate dehydrogenase complex

Two analogues of pyruvate, acetylphosphinate and acetylmethylphosphinate were tested as inhibitors of the E1 (pyruvate dehydrogenase) component of the human and Escherichia coli pyruvate dehydrogenase complexes. This is the first instance of such studies on the human enzyme. The acetylphosphinate is a stronger inhibitor of both enzymes (Ki < 1 microM) than acetylmethylphosphinate. Both inhibitors are found to be reversible tight-binding inhibitors. With both inhibitors and with both enzymes, the inhibition apparently takes place by formation of a C2alpha-phosphinolactylthiamin diphosphate derivative, a covalent adduct of the inhibitor and the coenzyme, mimicking the behavior of substrate and forming a stable analogue of the C2alpha-lactylthiamin diphosphate. Formation of the intermediate analogue in each case is confirmed by the appearance of a positive circular dichroism band in the 305-306 nm range, attributed to the 1',4'-iminopyrimidine tautomeric form of the coenzyme. It is further shown that the alphaHis63 residue of the human E1 has a role in the formation of C2alpha-lactylthiamin diphosphate since the alphaHis63Ala variant is only modestly inhibited by either inhibitor, nor did either compound generate the circular dichroism bands assigned to different tautomeric forms of the 4'-aminopyrimidine ring of the coenzyme seen with the wild-type enzyme. Interestingly, opposite enantiomers of the carboligase side product acetoin are produced by the human and bacterial enzymes.     

Cofactor activation and substrate binding in pyruvate decarboxylase. Insights into the reaction mechanism from molecular dynamics simulations

We have performed long-term molecular dynamics simulations of pyruvate decarboxylase from Zymomonas mobilis. Nine structures were modeled to investigate mechanistic questions related to binding of the cofactor, thiamin diphosphate (ThDP), and the substrate in the active site. The simulations reveal that the proposed three ThDP-tautomers all can bind in the active site and indicate that the equilibrium is shifted toward 4'-aminopyrimidine ThDP in the absence of substrate. 4'-Aminopyrimidinium ThDP is found to be a likely intermediate in the equilibrium. Mutations of important active site residues, Glu473Ala and Glu50Ala, were modeled to further elucidate their catalytic role. Formation of the catalytic important ylide by deprotonation of ThDP(C2) is investigated. Only the less favored tautomer, 1',4'-iminopyrimidine ThDP (imino-ThDP), could be deprotonated. The two other tautomers of ThDP could not be activated at the C2-position, thus, explaining the mechanistic importance of the less stable imino-ThDP. Finally, binding of pyruvate in the active site with the cofactor modeled as the nucleophilic ylide (ylide-ThDP) is studied. The carbonyl group of the substrate forms a hydrogen bond to Tyr290(OH). No hydrogen bond could be identified between ThDP(N4') and the substrate. The geometry of the substrate binding is well-suited for a nucleophilic attack by ylide-ThDP(C2). We propose that a proton relay from His113 via Asp27 and Tyr290 to the carbonyl oxygen atom of the substrate may be involved in the mechanism.     

Spectroscopic evidence for participation of the 1',4'-imino tautomer of thiamin diphosphate in catalysis by yeast pyruvate decarboxylase

The 1',4'-iminopyrimidine tautomeric form of the coenzyme thiamin diphosphate (ThDP), implicated in catalysis on the basis of the conformation of enzyme-bound ThDP, has been observed by both ultraviolet absorption and circular dichroism spectroscopy. On yeast pyruvate decarboxylase, the unusual tautomer is observed in an active center variant in which catalysis in the post-decarboxylation regime of the reaction is compromised. In a model system consisting of N1-methyl-4-aminopyrimidinium or N1-methyl-N4-n-butylpyrimidinium salts, on treatment with either NaOH in water, or DBU in DMSO there is an intermediate formed with lambda(max) near 310 nm, and this intermediate reverts back to the starting salt on acidification. Proton NMR chemical shifts are consistent with the intermediate representing the 1-methyl-4-imino tautomer. On the enzyme, the intermediate could be observed by rapid-scan stopped flow with UV detection when reacting holoenzyme of the E477Q active center variant with pyruvate, and by circular dichroism even in the absence of pyruvate. This represents the first direct observation of the imino tautomeric form of ThDP both on the enzyme and in models, although some years ago, this laboratory had already reported some pertinent acid-base properties for its formation [Jordan, F., and Mariam, Y. H. (1978) J. Am. Chem. Soc.100, 2534-2541]. The work also represents the first instance in which a rare tautomer implicated in catalysis is identified and suggests that such tautomeric catalysis may be more common in biology than hitherto recognized.     

Elucidation of the chemistry of enzyme-bound thiamin diphosphate prior to substrate binding: defining internal equilibria among tautomeric and ionization states

Both solution and crystallographic studies suggest that the 4'-aminopyrimidine ring of the thiamin diphosphate coenzyme participates in catalysis, likely as an intramolecular general acid-base catalyst via the unusual 1',4'-iminopyrimidine tautomer. It is indeed uncommon for a coenzyme to be identified in its rare tautomeric form on its reaction pathways, yet this has been possible with thiamin diphosphate, in some cases even in the absence of substrate [Nemeria, N., Chakraborty, S., Baykal, A., Korotchkina, L., Patel, M. S., and Jordan, F. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 78-82.]. The ability to detect both the aminopyrimidine and iminopyrimidine tautomeric forms of thiamin diphosphate on enzymes has enabled us to assign the predominant tautomeric form present in individual intermediates on the pathway. Herein, we report the pH dependence of these tautomeric forms providing the first data for the internal thermodynamic equilibria on thiamin diphosphate enzymes for the various ionization and tautomeric forms of this coenzyme on four enzymes: benzaldehyde lyase, benzoylformate decarboxylase, pyruvate oxidase, and the E1 component of the human pyruvate dehydrogenase multienzyme complex. Evidence is provided for an important function of the enzyme environment in altering both the ionization and tautomeric equilibria on the coenzyme even prior to addition of substrate. The pKa for the 4'-aminopyrimidinium moiety coincides with the pH for optimum activity thereby ensuring that all ionization states and tautomeric states are accessible during the catalytic cycle. The dramatic influence of the protein on the internal equilibria also points to conditions under which the long-elusive ylide intermediate could be stabilized.     

Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates. Evidence for reversible tight-binding inhibition

Variants of the pyruvate dehydrogenase subunit (E1; EC ) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. The K(m) values for thiamin diphosphate (ThDP) were 1.58 microm (parental E1) and 6.65 microm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 microm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr(177) substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K(i) = 0.003 microm for ThTDP and K(i) = 0.064 microm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr(177). Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr(177), the kinetic and spectroscopic data suggest that Tyr(177) has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in reductive acetylation of the dihydrolipoamide acetyltransferase component.     

Function of a conserved loop of the beta-domain, not involved in thiamin diphosphate binding, in catalysis and substrate activation in yeast pyruvate decarboxylase

The X-ray crystal structure of pyruvamide-activated yeast pyruvate decarboxylase (YPDC) revealed a flexible loop spanning residues 290 to 304 on the beta-domain of the enzyme, not seen in the absence of pyruvamide, a substrate activator surrogate. Site-directed mutagenesis studies revealed that residues on the loop affect the activity, with some residues reducing k(cat)/K(m) by at least 1000-fold. In the pyruvamide-activated form, the loop located on the beta domain can transfer information to the active center thiamin diphosphate (ThDP) located at the interface of the alpha and gamma domains. The sigmoidal v(0)-[S] curve with wild-type YPDC attributed to substrate activation is modulated for most variants, but is not abolished. Pre-steady-state stopped-flow studies for product formation on these loop variants provided evidence for three enzyme conformations connected by two transitions, as already noted for the wild-type YPDC at pH 5.0 [Sergienko, E. A., and Jordan, F. (2002) Biochemistry 41, 3952-3967]. (1)H NMR analysis of the intermediate distribution resulting from acid quench [Tittmann et al. (2003) Biochemistry 42, 7885-7891] with all YPDC variants indicated that product release is rate limiting in the steady state. Apparently, the loop is not solely responsible for the substrate activation behavior, rather it may affect the behavior of residue C221 identified as the trigger for substrate activation. The most important function of the loop is to control the conformational equilibrium between the "open" and "closed" conformations of the enzyme identified in the pyruvamide-activated structure [Lu et al. (2000) Eur. J. Biochem. 267, 861-868].     

Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects

Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of ( R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda max 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of ( R)-benzoin synthesis, the (1)H/ (2)H kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2alphaH from C2alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that while a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue ( E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product ( E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.     

Effect of thiamine phosphates on the activity of regulatory enzymes of the pyruvate dehydrogenase complex

The effect of thiamine triphosphate (ThTP) and thiamine diphosphate (ThDP) on the activity of rat liver pyruvate dehydrogenase complex regulatory enzymes (kinase and phosphatase) was studied in experiments with isolated enzyme preparations. It is shown that ThDP caused a pronounced activation of pyruvate dehydrogenase phosphatase (Ka is equal to 65.0 nM). ThTP inhibits phosphatase competitively against the substrate--the phosphorylated pyruvate dehydrogenase complex. The both thiamine phosphates inhibit the pyruvate dehydrogenase kinase activity almost similarly in concentrations exceeding 10 microM. The physiological significance of the antagonistic action of ThDP and ThTP on the pyruvate dehydrogenase phosphatase activity is discussed.     

Theoretical model of interactions between ligand-binding sites in a dimeric protein and its application for the analysis of thiamine diphosphate binding to yeast transketolase

The binding of thiamin diphosphate (ThDP) to yeast dimeric apotransketolase (apoTK) is accompanied by the appearance of a band in the absorption spectrum with maximum at 320 nm. The saturation function has been analyzed using a scheme that involves binding of ThDP to each subunit followed by the conformational transition of this subunit. It is assumed that the binding of ThDP to one subunit may affect the conformational transition of the other subunit. Rigorous mathematical expressions describing the dependence of the optical absorption on the total concentration of ThDP are first developed. Equilibrium constants and corresponding rate constants for the binding of ThDP to apoTK have been estimated. The negative cooperativity in the ThDP binding has been characterized by the function reflecting the dependence of the conformational change on the saturation of apoTK by ThDP.     

Structure-based rational design of novel hit compounds for pyruvate dehydrogenase multienzyme complex E1 components from Escherichia coli

Pyruvate dehydrogenase multienzyme complex (PDHc) E1 component plays a pivotal role in cellular metabolism to convert the product of glycolysis (pyruvate) to acetyl-CoA, and has been reported as a potential target for anti-microbial and herbicide. In present study, based on the thiamin diphosphate (ThDP) site, four novel hit compounds with high inhibitory activity against the PDHc-E1 from Escherichia coli were firstly designed by using structure-based molecular docking methods. As expected, among four compounds, the compound 3a is the best inhibitor by far, with IC₅₀ value of 6.88 μM against PDHc-E1 from E. coli. To elucidate the interaction mechanism between the active site of PDHc-E1 and its inhibitor, the docking-based molecular dynamics simulation (MD) and MD-based ab initio fragment molecular orbital (FMO) calculations were also further performed. The positive results indicated that all modeling strategies presented in the current study most like to be an encouraging way in design of novel lead compounds with structural diversity for PDHc-E1 in the future.     

Binding and activation of thiamin diphosphate in acetohydroxyacid synthase

Acetohydroxyacid synthases (AHASs) are biosynthetic thiamin diphosphate- (ThDP) and FAD-dependent enzymes. They are homologous to pyruvate oxidase and other members of a family of ThDP-dependent enzymes which catalyze reactions in which the first step is decarboxylation of a 2-ketoacid. AHAS catalyzes the condensation of the 2-carbon moiety, derived from the decarboxylation of pyruvate, with a second 2-ketoacid, to form acetolactate or acetohydroxybutyrate. A structural model for AHAS isozyme II (AHAS II) from Escherichia coli has been constructed on the basis of its homology with pyruvate oxidase from Lactobacillus plantarum (LpPOX). We describe here experiments which further test the model, and test whether the binding and activation of ThDP in AHAS involve the same structural elements and mechanism identified for homologous enzymes. Interaction of a conserved glutamate with the N1' of the ThDP aminopyrimidine moiety is involved in activation of the cofactor for proton exchange in several ThDP-dependent enzymes. In accord with this, the analogue N3'-pyridyl thiamin diphosphate does not support AHAS activity. Mutagenesis of Glu47, the putative conserved glutamate, decreases the rate of proton exchange at C-2 of bound ThDP by nearly 2 orders of magnitude and decreases the turnover rate for the mutants by about 10-fold. Mutant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of Asp428, presumed on the basis of the model to be the crucial carboxylate ligand to Mg(2+) in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg(2+). While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg(2+), D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K(+). These experiments demonstrate that AHAS binds and activates ThDP in the same way as do pyruvate decarboxylase, transketolase, and other ThDP-dependent enzymes. The biosynthetic activity of AHAS also involves many other factors beyond the binding and deprotonation of ThDP; changes in the ligands to ThDP can have interesting and unexpected effects on the reaction.     

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

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.     

Flexibility of thiamine diphosphate revealed by kinetic crystallographic studies of the reaction of pyruvate-ferredoxin oxidoreductase with pyruvate

Pyruvate-ferredoxin oxidoreductases (PFOR) are unique among thiamine pyrophosphate (ThDP)-containing enzymes in giving rise to a rather stable cofactor-based free-radical species upon the decarboxylation of their first substrate, pyruvate. We have obtained snapshots of unreacted and partially reacted (probably as a tetrahedral intermediate) pyruvate-PFOR complexes at different time intervals. We conclude that pyruvate decarboxylation involves very limited substrate-to-product movements but a significant displacement of the thiazolium moiety of ThDP. In this respect, PFOR seems to differ substantially from other ThDP-containing enzymes, such as transketolase and pyruvate decarboxylase. In addition, exposure of PFOR to oxygen in the presence of pyruvate results in significant inhibition of catalytic activity, both in solution and in the crystals. Examination of the crystal structure of inhibited PFOR suggests that the loss of activity results from oxime formation at the 4' amino substituent of the pyrimidine moiety of ThDP.     

Binding of the coenzyme and formation of the transketolase active center

Transketolase (TK) is a homodimer, the simplest representative of thiamine diphosphate (ThDP)-dependent enzymes. It was first ThDP-dependent enzymes the crystal structure of which has been solved and revealed the general fold for this class of enzymes and the interactions of the non-covalently bound coenzyme ThDP with the protein component. Transketolase is a convenient model to study the structure(s) of the active center and the mechanism of action of ThDP-dependent enzymes. This review summarizes the results of studies on the kinetics of the interaction of ThDP with TK from Saccharomyces cerevisiae as well as the generation of the catalytically active form of the coenzyme within the holoenzyme and formation of the enzyme's active center.     

Crystallographic snapshots of oxalyl-CoA decarboxylase give insights into catalysis by nonoxidative ThDP-dependent decarboxylases

Despite more than five decades of extensive studies of thiamin diphosphate (ThDP) enzymes, there remain many uncertainties as to how these enzymes achieve their rate enhancements. Here, we present a clear picture of catalysis for the simple nonoxidative decarboxylase, oxalyl-coenzyme A (CoA) decarboxylase, based on crystallographic snapshots along the catalytic cycle and kinetic data on active site mutants. First, we provide crystallographic evidence that, upon binding of oxalyl-CoA, the C-terminal 13 residues fold over the substrate, aligning the substrate alpha-carbon for attack by the ThDP-C2 atom. The second structure presented shows a covalent reaction intermediate after decarboxylation, interpreted as being nonplanar. Finally, the structure of a product complex is presented. In accordance with mutagenesis data, no side chains of the enzyme are implied to directly participate in proton transfer except the glutamic acid (Glu-56), which promotes formation of the 1',4'-iminopyrimidine tautomer of ThDP needed for activation.