Hydroxyethyl Thiamine Pyrophosphate Research Articles

The roles of coenzyme A in the pyruvate:ferredoxin oxidoreductase reaction mechanism: rate enhancement of electron transfer from a radical intermediate to an iron-sulfur cluster.

Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the coenzyme A (CoA)-dependent oxidative decarboxylation of pyruvate. In many autotrophic anaerobes, PFOR links the Wood-Ljungdahl pathway to glycolysis and to cell carbon synthesis. Herein, we cloned and sequenced the M. thermoacetica PFOR, demonstrating strong structural homology with the structurally characterized D. africanus PFOR, including the presence of three [4Fe-4S] clusters per monomeric unit. The PFOR reaction includes a hydroxyethyl-thiamin pyrophosphate (HE-TPP) radical intermediate, which forms rapidly after PFOR reacts with pyruvate. This step precedes electron transfer from the HE-TPP radical intermediate to an intramolecular [4Fe-4S] cluster. We show that CoA increases the rate of this redox reaction by 10(5)-fold. Analysis by Marcus theory indicates that, in the absence of CoA, this is a true electron-transfer reaction; however, in its presence, electron transfer is gated by an adiabatic event. Analysis by the Eyring equation indicates that entropic effects dominate this rate enhancement. Our results indicate that the energy of binding CoA contributes minimally to the rate increase since the thiol group of CoA lends over 40 kJ/mol to the reaction, whereas components of CoA that afford most of the cofactor's binding energy contribute minimally. Major conformational changes also do not appear to explain the rate enhancement. We propose several ways that CoA can accomplish this rate increase, including formation of a highly reducing adduct with the HE-TPP radical to increase the driving force for electron transfer. We also consider the possibility that CoA itself forms part of the electron-transfer pathway.

ENDOR Studies of Pyruvate:Ferredoxin Oxidoreductase Reaction Intermediates

Electron−nuclear double resonance (ENDOR) studies of radical intermediates formed by the oxidative decarboxylation of pyruvate by pyruvate:ferredoxin oxidoreductase were carried out to characterize their electronic structure and elucidate aspects of the recently proposed catalytic mechanism (Menon, S.; Ragsdale, S. W. Biochemistry 1997, 36, 8484−8494). The EPR spectrum of the PFOR/pyruvate adduct at 4 K displays a narrow resonance centered at g = 2.008 that has been attributed to a hydroxyethyl thiamine pyrophosphate (HE-TPP) radical. This spectral feature is superimposed on a broad, complex line shape characteristic of magnetically coupled [Fe4S4] clusters. The ENDOR spectrum at g = 2.008 reveals a broad peak with a complex line shape that can be analyzed, assuming that it arises from a composite of two axially symmetric proton hyperfine couplings. The principle coupling values for these two hyperfine tensors were: A∥(1) = 18.9 MHz, A⊥(1) = 12.6 MHz; and A∥(2) = 20.3 MHz, A⊥(2) = 14.9 MHz. The assignment of these features to the methyl protons of the pyruvate substrate was made using isotopic substitution. The temperature independence of these 1H ENDOR line shapes from 4 to 200 K indicates that the methyl group of pyruvate undergoes rapid rotation even at 4 K. The ENDOR spectrum at g = 2.008 also shows a pair of derivative peaks centered about the 31P Larmor frequency that are assigned to a weak hyperfine coupling with the phosphorus nuclei of the TPP cofactor. Two models for the electronic structure of the radical intermediate are discussed. A σ radical model which postulates a pyruvate-derived acetyl-type radical where little unpaired spin density resides on the TPP cofactor, and a π radical model that calls for more extensive delocalization of the unpaired electron spin over the HE-TPP framework. Both models require association of the radical center with the pyrophosphate group of TPP to interpret the observed 31P hyperfine coupling.

The presence of a hydroxyl group at the C-1 atom of the transketolase substrate molecule is necessary for the enzyme to perform the transferase reaction

Transketolase catalyzes the transfer of an aldehyde residue from keto sugars to aldo sugars. The intermediate product is dihydroxyethylthiamine pyrophosphate (DHETPP). In the absence of an acceptor substrate, the reaction is stopped at this stage and DHETPP does not undergo subsequent transformations. Pyruvate decarboxylase catalyses pyruvate decarboxylation to yield free aldehyde. The intermediate product is hydroxyethylthiamine pyrophosphate (HETPP). It differs from DHETPP only in that it has no hydroxyl at the C-2 atom of the aldehyde residue. We have shown that transketolase can bind HETPP and split the aldehyde residue from it. This fact suggests that the path of the reaction is determined by the absence (in HETPP) or presence (in DHETPP) of a hydroxyl group. In the former case the reaction will yield free aldehyde, in the latter the aldehyde residue will be transferred onto an acceptor substrate.

Activation of Escherichia coli pyruvate oxidase enhances the oxidation of hydroxyethylthiamin pyrophosphate

The catalytic efficiency (kcat/Km) of Escherichia coli flavin pyruvate oxidase can be stimulated 450-fold either by the addition of lipid activators or by limited proteolytic hydrolysis. Previous studies have shown that a functional lipid binding site is a mandatory prerequisite for the in vivo functioning of this enzyme (Grabau, C., and Cronan, J. E., Jr. (1986) Biochemistry 25, 3748-3751). The effect of activation on the transient state kinetics of partial reactions in the overall oxidative conversion of pyruvate to acetate and CO2 has now been examined. The rate of decarboxylation of pyruvate to form CO2 and hydroxyethylthiamin pyrophosphate for both activated and unactivated forms of the enzyme is identical within experimental error. The decarboxylation step was measured using substrate concentrations of the enzyme in the absence of an electron acceptor. The pseudo-first order rate constant for the decarboxylation step is 60-80 s-1. The rate of oxidation of hydroxyethylthiamin pyrophosphate and concomitant enzyme-bound flavin reduction was analyzed by stopped-flow methods utilizing synthetic hydroxyethylthiamin pyrophosphate. The pseudo-first order rate for this step with unactivated enzyme was 2.85 s-1 and increased 145-fold for lipid-activated enzyme to 413 s-1 and 61-fold for the proteolytically activated enzyme to 173 s-1. The analysis of a third reaction step, the reoxidation of enzyme-bound FADH, was also investigated by stopped-flow techniques utilizing ferricyanide as the electron acceptor. The rate of oxidation of enzyme.FADH is very fast for both unactivated (1041 s-1) and activated enzyme (645 s-1). The data indicate that the FAD reduction step is the rate-limiting step in the overall reaction for unactivated enzyme. Alternatively, the rate-limiting step in the overall reaction with the activated enzyme shifts to one of the partial steps in the decarboxylation reaction.

Mechanism of pyruvate inhibition of plant pyruvate dehydrogenase kinase and synergism with ADP

The effects of various metabolites on pyruvate dehydrogenase (PDH) kinase-catalyzed inactivation of the pyruvate dehydrogenase complex (PDC) were studied in extracts of mitochondria purified from green leaf tissue of Pisum sativum L. Pyruvate was an uncompetitive inhibitor of PDH kinase with respect to ATP whereas ADP was a competitive inhibitor. In the absence of pyruvate a fivefold excess of ADP over ATP was required to inhibit PDH kinase, however, in the presence of pyruvate much lower ADP concentrations were required. Inhibition of PDH kinase by pyruvate and ADP was synergistic and the addition of ADP changed pyruvate from an uncompetitive inhibitor to a noncompetitive inhibitor. This result indicates that pyruvate acts as a “dead-end” inhibitor, binding to the PDH kinase—ADP reaction intermediate. Evidence is also presented that inhibition by pyruvate in the presence of thiamine pyrophosphate is due to the formation of hydroxyethyl thiamine pyrophosphate. The results are discussed in terms of the regulation of PDC activity by pyruvate and ADP during periods of increased demand for carbon skeleton biosynthesis by way of the tricarboxylic acid (TCA) cycle despite constraints imposed on TCA cycle flux by a high ATP/ADP ratio.

Spin-label studies on the lipoamide residues of the pyruvate dehydrogenase multienzyme complex of escherichia coli

The catalytic mechanism of the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been investigated using spin-labelling and synthetic inhibitors of the enzyme. The lipoamide residues covalently bound to the lipote acetyltransferase component of the complex were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl in the presence of pyruvate. The electron spin resonance spectrum of the spin-labelled enzyme displayed two components, one highly mobile (correlation time about 1 ns) and the other immobilized (correlation time much greater than 50 ns). The temperature-dependence of the signal intensities of the two components suggested that the lipoamide residues can occupy two alternative states, the proportion of spin-labeled lipaomide in the mobile state increasing as the temperature is raised. Pyruvate caused a fall in the intensity of the ESR signal from the spin-labelled lipaomide residues in the presence of thiamine pyrophosphate and Mg 2+. Acetoin also led to loss of ESR signal, but, with both pyruvate and acetoin, the additio of thiamine thiazolone pyprophosphate prevented the loss of signal. The rate of loss of ESR signal intensity was consistent with an intramolecular reaction between lipaomide-bound nitroxide groups and hydroxyethylthiamine pyrophosphate. The similar rates of reduction by isoascorbate of nitroxide spin-lebel free in solution and bound to the lipoamide residues of the pyruvate dehydrogenase complex indicated free access of the isoascorbate to the enzyme-boynd nitroxide groups. 13P-NMR of acetyl phosphonate, a competitive inhibitor of the enzyme, bound to native enzyme and to spin-labelled enzyme, showed a large chemical shift on binding, but the linewidths did not indicate any spin-spin interaction between the nitroxide radicals and the 31P nuclei. These results agree with recent experimental data from kinetic studies and 1H-NMR spectroscopy on the system.

Studies of the flavin adenine dinucleotide binding region in Escherichia coli pyruvate oxidase.

Experiments have been performed to probe the flavin adenine dinucleotide (FAD) binding region in Escherichia coli pyruvate oxidase. This enzyme functions as a membrane-associated flavoprotein coupled to the aerobic E. coli respiratory chain. The FAD moiety is noncovalently bound to pyruvate oxidase and can be removed reversibly to form apopyruvate oxidase. The addition of free FAD to apoenzyme results in the stoichiometric re-formation of the active flavoprotein. Using this technique, synthetic analogs of FAD were substituted in the flavin binding site and used as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-hydroxy-FAD are stabilized in the enzyme-binding site. This is consistent with the fact that the native flavoprotein forms a red (anion) radical upon photoreduction. These data suggest that the isoalloxazine ring may be poised for reduction via position N-5 by a carbanionic intermediate. The alpha-carbanion of hydroxyethylthiamin pyrophosphate, formed following the decarboxylation of pyruvate, is a likely candidate. The highly resolved visible spectrum of the native flavoprotein suggests that the flavin is buried in a hydrophobic environment. Reactivity studies using 8-chloro-FAD-pyruvate oxidase and 2-thio-FAD-pyruvate oxidase suggest that the C-8 position and C-2 position of the isoalloxazine ring may not be accessible to the solvent. Spectral perturbations observed with 6-hydroxy-FAD-pyruvate oxidase indicate, however, that the isoalloxazine C-6 position may be located near the binding site for the cofactor thiamin pyrophosphate. Restrictions to the accessibility of the active site of the enzyme are suggested by the fact that sulfite does not form an adduct with the flavin in the native enzyme.

Phosphonate analogues of pyruvate. Probes of substrate binding to pyruvate oxidase and other thiamin pyrophosphate-dependent decarboxylases

A number of enzymes catalyze the removal of carbon dioxide from pyruvate through covalent participation of the coenzyme thiamin pyrophosphate. The conversions of the decarboxylated adduct, hydroxyethyl thiamin pyrophosphate, to subsequent products distinguishes the function of these enzymes. Acetaldehyde is produced by pyruvate decarboxylase, acetic acid by pyruvate oxidase and acetyl coenzyme A by pyruvate dehydrogenase. Differences and details of steps prior to decomposition of hydroxyethyl thiamin pyrophosphate can be evaluated through the use of two substrate analogues, methyl acetylphosphonate and acetylphosphonate. Methyl acetylphosphonate and acetylphosphonate are competitive inhibitors toward pyruvate with Escherichia coli pyruvate oxidase and E. coli pyruvate dehydrogenase but the value of the K i for the oxidase is more than three orders of magnitude higher than for the dehydrogenase. Yeast pyruvate decarboxylase is not inhibited at all under the same conditions. The binding of methyl acetylphosphonate results in ligand-induced changes in the near ultraviolet circular dichorism spectrum of the oxidase. This spectral perturbation is only seen in the presence of the cofactor, thiamin pyrophosphate, strongly suggesting that the inhibitor is binding at the same site as the substrate, pyruvate, on the enzyme. Kinetic data suggest that lipid activators of pyruvate oxidase increase the affinity of the enzyme for pyruvate and its analogues.

Profiles in altered metabolism I--the organic acids accumulating in acute non-diabetic ketoacidosis associated with alcoholism.

Several organic acids, among them the acidic catabolites of the branched chain amino acids and tyrosine, have been found to be elevated in the sera of non-diabetic patients presenting acute ketoacidosis associated with alcohol abuse. These findings are interpreted in terms of insufficiency of dietary co-factors required for their further catabolism. Butane-2,3-diol is also found frequently elevated in the urine of these patients and suggests interception of hydroxyethyl thiamine pyrophosphate by circulating high levels of acetaldehyde.

Biosynthesis of alkane-2,3-diols: Chemical synthesis of 3-hydroxy-[3- 14C] octadecane-2-one and its reduction to [3- 14C]octadecane-2,3-diol in the uropygial glands of ring-necked pheasants ( Phasianus colchicus)

Acyloin has been proposed to be an intermediate in the biosynthesis of long chain alkane-2,3-diols. In order to test this possibility, specifically labeled 3-hydroxyoctadecane-2-one (acyloin) was synthesized by coupling 2-methyl-1,3-dithiane with [1- 14C]hexadecanal followed by cleaving of the thioketal. Injection of the synthetic 3-hydroxy [3- 14C]octadecane-2-one into the uropygial gland of the ring-necked pheasant resulted in the formation of labeled octadecane-2,3-diol. Chemical degradation of this diol showed that all of the 14C was contained in C-3 of the diol showing direct conversion of acyloin to the diol. These observations support the hypothesis that alkane-2,3-diols might be biosynthesized by reduction of the acyloin derived from a condensation between hydroxyethyl thiamine pyrophosphate and fatty aldehyde. Gas-liquid chromatographic analysis of the alkane-2,3-diols, as their isopropylidene derivatives, of the pheasant strongly suggests that they are of the erythro-configuration; however, alkane-2,3-diol enzymatically formed from the racemic acyloin injected into the gland contained 59.5% erythro- and 40.5% threo-diastereoisomers. This distribution was identical to that produced by chemical reduction of the synthetic racemic acyloin. These results clearly show that the reduction step does not show a preference for either of the enantiomers of the acyloin and that the stereospecificity in diol biosynthesis probably resides in the condensation step.

Acetoin degradation in Bacillus subtilis by direct oxidative cleavage.

Acetate and acetaldehyde can be detected as products of the oxidative dissimilation of acetoin in Bacillus subtilis extracts. They arise as the result of the direct cleavage of acetoin without a previous oxidation to diacetyl. This can be deduced from the following observations: (a) no diacetyl was detected in acetoin dissimilation experiments in vitro and (b) methylacetoin, an acetoin analogue which can not be oxidized to the diketone, also undergoes oxidative splitting, yielding acetone and acetate. The splitting reaction requires thiamine pyrophosphate as a cofactor, suggesting that the oxidative step occurs, as known for similar reactions, by the electron transfer from hydroxyethylthiamine pyrophosphate to a proper acceptor, which in vitro can be replaced by dichlorophenolindophenol. In vivo the final product of the oxidation of hydroxyethylthiamine pyrophosphate is activated acetate. A mutant which lacks acetoin-cleaving activity can not reutilize the acetoin accumulated after growth in glucose. This corroborates the actual importance of the cleavage reaction for acetoin dissimilation. The enzyme diacetylmethylcarbinol synthase, thought to be responsible for the formation of diacetylmethylcarbinol from diacetyl, probably is identical to the enzyme catalyzing the cleavage of acetoin.

Influence of Acetaldehyde on Growth and Acetoin Production by Leuconostoc citrovorum

Addition of acetaldehyde (100μg/ml) to glucose-citrate broth stimulated growth and approximately doubled the production of acetoin plus diacetyl by Leuconostoc citrovorum. Radioactive acetaldehyde was converted to ethanol; the acetoin, diacetyl and acetic acid produced were not radioactive. A discussion of the mechanisms by which species of Leuconostoc utilize citrate and carbohydrate suggests that the added acetaldehyde enhanced production of acetoin and diacetyl by increasing the availability of hydroxyethylthiamine pyrophosphate and acetyl-coenzyme A, and that it stimulated growth by permitting greater conversion of acetyl-coenzyme A and acetyl-phosphate to acetate and adenosine 5′-triphosphate.

Incorporation of radioactive acetate into diacetyl by Streptococcus diacetilactis.

Streptococcus diacetilactis was grown in a partially defined, lipoic acid-free medium containing radioactive acetate with and without addition of 0.1% unlabeled sodium pyruvate. Labeled carbon was incorporated into diacetyl, but neither the amount of diacetyl produced nor its specific activity was influenced by addition of pyruvate. Acetoin had low specific activity, indicating that it was a mixture of radioactive and nonradioactive acetoin. The specific activity of acetoin was lower when pyruvate, a precursor of unlabeled acetoin, was added to the medium, which indicated that the radioactive acetoin was produced from radioactive diacetyl by diacetyl reductase. Results substantiate condensation of acetyl-coenzyme A with hydroxyethylthiamine pyrophosphate as the in vivo mechanism for synthesis of diacetyl.

Incorporation of Radioactive Acetate into Diacetyl by Streptococcus diacetilactis

Streptococcus diacetilactis was grown in a partially defined, lipoic acid-free medium containing radioactive acetate with and without addition of 0.1% unlabeled sodium pyruvate. Labeled carbon was incorporated into diacetyl, but neither the amount of diacetyl produced nor its specific activity was influenced by addition of pyruvate. Acetoin had low specific activity, indicating that it was a mixture of radioactive and nonradioactive acetoin. The specific activity of acetoin was lower when pyruvate, a precursor of unlabeled acetoin, was added to the medium, which indicated that the radioactive acetoin was produced from radioactive diacetyl by diacetyl reductase. Results substantiate condensation of acetyl-coenzyme A with hydroxyethylthiamine pyrophosphate as the in vivo mechanism for synthesis of diacetyl.

Studies on the biosynthesis of the branched-chain sugars from the quinocycline complex.

The incorporation of [2‐14C]pyruvate and [1‐14C]acetate into the 2,6‐dideoxy‐4‐C‐[1′‐hydroxy‐ethyl]‐l‐xylo‐hexopyranose (compound I) moiety of quinocycline A and into the 2,6‐dideoxy‐4‐C‐[1′‐oxoethyl]‐l‐xylo‐hexopyranose (compound II) portion of quinocycline B produced by Streptomyces aureofaciens was investigated. The methylglycosides of compounds I and II were subjected to periodate oxidation to yield, respectively, acetaldehyde and acetic acid from the branch carbon atoms. Acetaldehyde was oxidized to acetic acid, which was then degraded by Schmidt‐degradation. With [2‐14C]pyruvate 87 to 90% of the radioactivity incorporated into compound I or II was located in the C2 branch without randomization of the label. In contrast, with [1‐14C]acetate only very low activity was incorporated into the sugars and no 14C was localized in the C2 branch. These results prove that the C2 branch in compounds I and II originates from C‐2 and C‐3 of pyruvic acid.When 14C‐labelled quinocycline B was incubated for 17 h with S. aureofaciens, 13% was converted to quinocycline A. Under the same conditions only 2% of [14C]quinocycline A was converted to quinocycline B. It can therefore be concluded that quinocycline B is the precursor of quinocycline A. This result is in agreement with the assumption that hydroxyethylthiamine pyrophosphate is the immediate precursor for the oxoethyl group of the branched sugar II, which after transfer to the aglycone is reduced to compound I. Experiments with the thiamine antagonist neopyrithiamine did not show inhibition of incorporation of radioactivity from [2‐14C]‐pyruvate into the hydroxyethyl branch of compound I. However, this result does not exclude the possibility that thiamine pyrophosphate participates in the reaction.

Pyruvate-Ferredoxin Oxidoreductase: IV. STUDIES ON THE REACTION MECHANISM

The sequence of the reaction catalyzed by pyruvate-ferredoxin oxidoreductase was investigated. The pyruvate14CO2 exchange reaction and acetoin formation are inhibited by electron acceptors such as ferredoxin, FAD, FMN, and dyes, but not by DPN. The enzyme catalyzes the formation of acetoin in the presence of pyruvate and acetaldehyde, which suggests that hydroxyethyl thiamine pyrophosphate is an intermediate of the reaction. When substrate levels of the enzyme reacted with pyruvate, a stoichiometric amount of HCO3- was formed but the enzyme chromophore is not reduced. The addition of coenzyme A to the reaction mixture in the absence of an electron acceptor leads to bleaching of the enzyme chromophore and formation of a stoichiometric amount of acetyl-CoA. The enzyme chromophore reduced by addition of pyruvate and CoA is reoxidized by FAD or ferredoxin. The chromophore is also reduced by reduced ferredoxin formed by the hydrogenase system. Based on these observations a reaction sequence is proposed and evidence in support of the mechanism is discussed. It is concluded that the enzyme chromophore, rather than ferredoxin, serves as a primary electron acceptor.