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Frank Jordan

Publications and source records attributed to Frank Jordan.

At least 19 recordsLinked to original sources
PubMed2006

The 1',4'-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes.

Thiamin diphosphate, a key coenzyme in sugar metabolism, is comprised of the thiazolium and 4'-aminopyrimidine aromatic rings, but only recently has participation of the 4'-aminopyrimidine moiety in catalysis gained wider acceptance. We report the use of electronic spectroscopy to identify the various tautomeric forms of the 4'-aminopyrimidine ring on four thiamin diphosphate enzymes, all of which decarboxylate pyruvate: the E1 component of human pyruvate dehydrogenase complex, the E1 subunit of Escherichia coli pyruvate dehydrogenase complex, yeast pyruvate decarboxylase, and pyruvate oxidase from Lactobacillus plantarum. It is shown that, according to circular dichroism spectroscopy, both the 1',4'-iminopyrimidine and the 4'-aminopyrimidine tautomers coexist on the E1 component of human pyruvate dehydrogenase complex and pyruvate oxidase. Because both tautomers are seen simultaneously, these two enzymes provide excellent evidence for nonidentical active centers (asymmetry) in solution in these multimeric enzymes. Asymmetry of active centers can also be induced upon addition of acetylphosphinate, an excellent electrostatic pyruvate mimic, which participates in an enzyme-catalyzed addition to form a stable adduct, resembling the common predecarboxylation thiamin-bound intermediate, which exists in its 1',4'-iminopyrimidine form. The identification of the 1',4'-iminopyrimidine tautomer on four enzymes is almost certainly applicable to all thiamin diphosphate enzymes: this tautomer is the intramolecular trigger to generate the reactive ylide/carbene at the thiazolium C2 position in the first fundamental step of thiamin catalysis.

Binding Sites↗
PubMed2006

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].

Amino Acid Sequence↗
PubMed2006

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.

Acetone↗
PubMed2006

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.

Circular Dichroism↗
PubMed2006

Active-site changes in the pyruvate dehydrogenase multienzyme complex E1 apoenzyme component from Escherichia coli observed at 2.32 A resolution.

The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase multienzyme complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg(2+) as cofactors. The structure of a branched-chain-specific E1 apoenzyme from the heterotetrameric alpha(2)beta(2) E1 family was recently reported and showed that disorder-to-order transformations in two active-site loops take place upon cofactor binding. To ascertain what effect the absence of cofactor may have in the homodimeric alpha(2) Escherichia coli PDHc E1, the corresponding apoenzyme has been prepared and its three-dimensional structure determined and analyzed at 2.32 A by crystallographic methods. This represents the first reported apoenzyme structure for any E1 component from the homodimeric alpha(2) family. Electron-density features occurring in the region where the cofactor pyrimidine ring would normally be expected to bind are of size, shape and location compatible with water molecules that form a hydrogen-bonded linkage between residues Glu571 and Val192, which normally make conserved interactions with the ThDP cofactor. A histidine side chain that normally forms hydrogen bonds to ThDP is disordered in its absence and partially occupies two sites. Unlike in the reported heterotetrameric branched-chain apo-E1, no disorder/order loop transformations are evident in apo-PDHc E1 relative to the holo-E1 enzyme (PDHc E1-ThDP-Mg(2+)). Differences in the extent of hydrogen-bonding networks found in the apo-E1 enzyme, the holo-E1 enzyme and in an inhibitor complex with bound thiamine 2-thiazolone diphosphate (ThTDP), PDHc E1-ThTDP-Mg(2+), are described.

Apoenzymes↗
PubMed2006

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.

Binding Sites↗
PubMed2006

A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

Amino Acid Substitution↗
PubMed2005

Experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations.

Thiamin diphosphate (ThDP), the vitamin B1 coenzyme, is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/C2-carbanion of the thiazolium ring and the C2alpha-carbanion (or enamine) once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state (X-ray) methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of a several intermediates which are stable under acidic conditions, and circular dichroism detection of the 1',4'-imino tautomer of ThDP in some of the intermediates. Some of these methods also enable the investigator to determine the rate-limiting step in the complex series of steps.

Coenzymes↗
PubMed2005

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.

Acetolactate Synthase↗
PubMed2005

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.

Acetylation↗
PubMed2005

Multiple modes of active center communication in thiamin diphosphate-dependent enzymes.

Detection of interaction between cofactors at the active centers of homodimeric and homotetrameric enzymes is usually elusive by steady-state kinetic approaches and requires protein variants where such interactions are diminished or exaggerated. In this Account, evidence for active-center interactions will be presented for the following thiamin diphosphate-dependent enzymes: yeast pyruvate decarboxylase, benzoylformate decarboxylase, and examples from the 2-oxoacid dehydrogenase multienzyme complex class. The dissymmetry of active sites is especially evident in the X-ray structures of these enzymes with substrate/substrate analogues bound. Perturbations that reveal active center communication include use of chromophoric substrates and substitutions of active center residues on putative pathways.

Binding Sites↗
PubMed2005

Improving removal-based estimates of abundance by sampling a population of spatially distinct subpopulations.

A statistical modeling framework is described for estimating the abundances of spatially distinct subpopulations of animals surveyed using removal sampling. To illustrate this framework, hierarchical models are developed using the Poisson and negative-binomial distributions to model variation in abundance among subpopulations and using the beta distribution to model variation in capture probabilities. These models are fitted to the removal counts observed in a survey of a federally endangered fish species. The resulting estimates of abundance have similar or better precision than those computed using the conventional approach of analyzing the removal counts of each subpopulation separately. Extension of the hierarchical models to include spatial covariates of abundance is straightforward and may be used to identify important features of an animal's habitat or to predict the abundance of animals at unsampled locations.

Animals↗
PubMed2004

Amino-terminal residues 1-45 of the Escherichia coli pyruvate dehydrogenase complex E1 subunit interact with the E2 subunit and are required for activity of the complex but not for reductive acetylation of the E2 subunit.

While N-terminal amino acids 1-55 are not seen in the structure of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), mass spectrometric analysis indicated that this amino-terminal region of PDHc-E1 was protected by PDHc-E2. Hence, five deletion constructs of PDHc-E1 were created, Delta6-15, Delta16-25, Delta26-35, Delta36-45, and Delta46-55, along with single-site substitutions at Asp7, Asp9, Pro10, Ile11, Glu12, Thr13, Arg14, and Asp15. The decarboxylation of pyruvate and the ability of PDHc-E1 to dimerize are not affected by any of the deletions or substitutions. While Delta46-55 and the Pro10Ala, Ile11Ala, and Thr13Ala variants could form a complex with PDHc-E2, and produced NADH in the overall assay, Delta16-25, Delta26-35, and Delta36-45 and the Asp7Ala, Asp9Ala, Glu12Gln, Glu12Asp, Arg14Ala, and Asp15Ala variants failed in both respects. Remarkably, all constructs of PDHc-E1 from E. coli, as well as PDHc-E1 from Mycobacterium tuberculosis, could carry out reductive acetylation of the E. coli lipoyl domain, but only constructs of the E. coli PDHc-E1 could reductively acetylate E. coli PDHc-E2. It was concluded that there are at least two loci of interaction between the PDHc-E1 and PDHc-E2 subunits: (1) the thiamin diphosphate-bound substrate on PDHc-E1 and the lipoylamide of PDHc-E2, as reflected by the ability to reductively acetylate the latter; and (2) amino terminal residues 1-45 of PDHc-E1 with regions of PDHc-E2 (so far undefined for the E. coli complex), as reflected by the overall activity of the entire complex. These studies add important information regarding recognition within this multienzyme complex class with an alpha(2) E1 assembly.

Acetylation↗
PubMed2004

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.

Acetaldehyde↗
PubMed2004

Tetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1',4'-imino tautomeric form of the coenzyme, unlike the michaelis complex or the free coenzyme.

Two circular dichroism signals observed on thiamin diphosphate (ThDP)-dependent enzymes, a positive band in the 300-305 nm range and a negative one in the 320-330 nm range, were investigated on yeast pyruvate decarboxylase (YPDC) and on the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1). Addition of the tetrahedral ThDP-acetaldehyde adduct, 2-alpha-hydroxyethylThDP, to PDHc-E1 generates the positive band at 300 nm, consistent with the formation of the 1',4'-iminopyrimidine tautomer, as also demonstrated for phosphonolactylthiamin diphosphate, a stable analogue of the tetrahedral ThDP-pyruvate adduct 2-alpha-lactylThDP (Jordan, F. et al. (2003) J. Am. Chem. Soc. 125, 12732-12738). Therefore, we suggest that all tetrahedral ThDP-bound covalent complexes will also prefer this tautomer, and that the 4'-aminopyrimidine of ThDP participates in multiple steps of acid-base catalysis on ThDP enzymes. Studies with YPDC and PDHc-E1, and their active center variants, in conjunction with chemical models, enabled assignment of the negative band at 330 nm to a charge-transfer transition between the 4'-aminopyrimidine tautomer (presumed electron donor) and the thiazolium ring (presumed electron acceptor) of ThDP, with no significant contributions from any amino acid side chain of the proteins. However, in both YPDC and PDHc-E1, the presence of substrate or substrate surrogate was required to enable detection, suggesting that the band at 320-330 nm be used as a reporter for the Michaelis complex, involving the amino tautomer, on both enzymes. As the positive band near 300 nm reports on the 1',4'-imino tautomer of ThDP, methods are now available for kinetic monitoring of both tautomeric forms.

Binding Sites↗
PubMed2004

Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

Binding Sites↗
PubMed2003

Dual catalytic apparatus of the thiamin diphosphate coenzyme: acid-base via the 1',4'-iminopyrimidine tautomer along with its electrophilic role.

It was recently reported (Jordan, F.; Zhang, Z.; Sergienko, E. A. Bioorg. Chem. 2002, 30, 188-198) that addition to the E477Q active-center variant of yeast pyruvate decarboxylase of (a) pyruvate on a rapid-scan UV stopped-flow, or (b) acetaldehyde or benzoylformate on a circular dichroism (CD) instrument, generates a new band with lambda(max) near 300-310 nm. A chemical model demonstrated that the wavelength is appropriate to the 1',4'-iminopyrimidine tautomer of the 4'-aminopyrimidine ring in thiamin diphosphate. Herein, we report the formation of a new positive CD band centered at 305 nm when the Escherichia colipyruvate dehydrogenase complex first E1 subunit and its variants are exposed to phosphonolactylthiamin diphosphate, a stable analogue of the covalent adduct formed between the substrate pyruvate and the C2 atom of thiamin diphosphate. The behavior of this CD band, whether it suggests saturation of the enzyme by phosphonolactylthiamin diphosphate, or its very existence (the band is not seen with the E571A E1 variant, where E571 is hydrogen bonded to the N1' atom of the 4'-aminopyrimidine ring), as well as its position are consistent with its assignment to the 1',4'-imino thiamin diphosphate tautomer on the enzyme, chiral by virtue of its fixed V conformation. The mechanism of binding of phosphonolactylthiamin diphosphate closely resembles that of thiamin diphosphate itself.

Binding Sites↗