Optimization of hydroxythiamine administration regimen in vitaminological research

A correlation between the different doses and inhibitory effect of hydroxythiamine relative to the activity of main thiamine pyrophosphate-dependent enzymes is studied in experiments on animals. It is established that the maximal inhibitory effect on the transketolase and oxoglutarate dehydrogenase activities is at a dose of antivitamin of 0.35 mmol/kg and that of pyruvate dehydrogenase of 0.55 mmol/kg. At lower doses of hydroxythiamine the inhibition of enzyme activities occurred in a dose-dependent manner.     

Enzyme activity of thiamine pyrophosphate in the rat after oxythiamine administration

Intraperitoneal injection of hydroxythiamine to rats (1 mmol per kg bw) resulted after 2-4 h in a more than 4-fold decrease in the activity of the oxoglutarate dehydrogenase complex, pyruvate dehydrogenase complex and NADP-dependent isocitrate dehydrogenase in adrenal mitochondria. Inhibition of hyaloplasmic transketolase, 6-phosphogluconate dehydrogenase and NADP-dependent malate dehydrogenase occurred later. Based on the correlation of the time course of enzymatic activity in the adrenals and the decreased concentration of 11-hydroxycorticosteroids in the blood the paramount role in the maintenance of the steroidogenesis among thiamine pyrophosphate-containing enzymes is assigned to the oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes.     

Kinetic Studies of Mouse Brain Transketolase

Abstract: The activity of transketolase in mouse brain was 5.7 nmol/min/mg protein measured by an enzyme-coupled spectrophotometric assay. The apparent K_m for ribose-5-phosphate was 330 μ M , for d -xylulose-5-phosphate was 120 μ M , and for thiamine pyrophosphate was 7 μ M . However, thiamine pyrophosphate remained tightly bound to transketolase in homogenates in which it dissociated completely from another thiamine pyrophosphate- dependent enzyme, the pyruvate dehydrogenase complex. These data suggest that loss of transketolase activity is likely to be a later consequence of thiamine deficiency in mammalian brain than is decreased activity of pyruvate dehydrogenase complex.     

Features of thiamine diphosphate-dependent enzyme inhibition by oxodihydrothiochrome and its phosphorylated derivatives

Anticoenzyme action of new derivatives of thiamine: oxodihydrothiochrome and its mono- and diphosphoric esters has been studied in the experiments on mice. It is shown that the given compounds exert an inhibiting action on transketolase and pyruvate dehydrogenase and do not change activity of 2-oxoglutarate dehydrogenase in the animal organism. Antivitamin effect of the studied inhibitors is observed with the lower doses and in the earlier terms as compared with the other known inhibitors of thiamine-diphosphate-dependent enzymes. The preparations inhibit activity of the yeast pyruvate-decarboxylase by the mixed (with respect to thiamine-diphosphate) type (Ki for oxodihydrothiochrome and its mono- and diphosphoric esters: 2.3 x 10(-3), 7.2 x 10(-4), 5.6 x 10(-5) M, respectively). Possible mechanisms of the action of the mentioned compounds as thiamine antimetabolites are discussed.     

Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.     

Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells

Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic 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.     

Correlation of enzymatic,metabolic, and behavioral deficits in thiamin deficiency and its reversal

To clarify the enzymatic mechanisms of brain damage inthiamin deficiency, glucose oxidation, acetylcholine synthesis, and the activities of the three major thiamin pyrophosphate (TPP) dependent brain enzymes were compared in untreated controls, in symptomatic pyrithiamin-induced thiamin-deficient rats, and in animals in which the symptoms had been reversed by treatment with thiamin. Although brain slices from symptomatic animals produced¹⁴CO₂ and¹⁴C-acetylcholine from [U-¹⁴C]glucose at rates similar to controls under resting conditions, their K⁺-induced-increase declined by 50 and 75%, respectively. In brain homogenates from these same animals, the activities of two TPP-dependent enzymes transketolase (EC 2.2.1.1) and 2-oxoglutarate dehydrogenase complex (EC 1.2.4.2, EC 2.3.1.61, EC 1.6.4.3) decreased 60–65% and 36%, respectively. The activity of the third TPP-dependent enzyme, pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.6.4.3.) did not change nor did the activity of its activator pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43). Although treatment with thiamin for seven days reversed the neurological symptoms and restored glucose oxidation, acetylcholine synthesis and 2-oxoglutarate dehydrogenase activity to normal, transketolase activity remained 30–32% lower than controls. The activities of other TPP-independent enzymes (hexokinase, phosphofructokinase, and glutamate dehydrogenase) were normal in both deficient and reversed animals.Thus, changes in the neurological signs during pyrithiamin-induced thiamin deficiency and in recovery paralleled the reversible damage to a mitochondrial enzyme and impairment of glucose oxidation and acetylcholine synthesis. A more sustained deficit in the pentose pathway enzyme, transketolase, may relate to the anatomical abnormalities that accompany thiamin deficiency.Dedicated to Henry McIlwain.     

Study on the role of SH-groups in the activity of muscle pyruvate dehydrogenase

The kinetics of inactivation of the pyruvate dehydrogenase component of the pigeon breast muscle pyruvate dehydrogenase complex in the presence of 5,5'-dithiobis (2-nitrobenzoate) is biphasic. The rate constants for the fast and slow phases of the inactivation reaction are close to those for modification of two classes of SH-groups differing in their reactivities towards the inhibitor. The reaction order with respect to the inhibitor concentration suggests that the two distinct SH-groups are essential for the enzyme activity. Modification of these SH-groups results in inhibition of the overall activity of the pyruvate dehydrogenase complex and of the 2-hydroxyethyl thiamine pyrophosphate - acceptor oxidoreductase activity of its decarboxylating component. Thiamine pyrophosphate exerts a protective effect on the enzyme only at the slow phase of the enzyme inactivation and SH-modification. As a result of interaction between the holoenzyme and pyruvate (or apoenzyme and 2-hydroxyethyl thiamine pyrophosphate) the rate of the enzyme inactivation is increased. This is associated with masking of non-essential SH-groups and with an increase of the accessibility of two essential SH-groups to the inhibitor. The data obtained suggest the interrelationship between the essential SH-groups and the 2-hydroxyethyl thiamine pyrophosphate-acceptor oxidoreductase activity of pyruvate dehydrogenase.     

Structure and functioning mechanism of transketolase

Studies of thiamine diphosphate-dependent enzymes appear to have commenced in 1937, with the isolation of the coenzyme of yeast pyruvate decarboxylase, which was demonstrated to be a diphosphoric ester of thiamine. For quite a long time, these studies were largely focused on enzymes decarboxylating α-keto acids, such as pyruvate decarboxylase and pyruvate dehydrogenase complexes. Transketolase, discovered independently by Racker and Horecker in 1953 (and named by Racker) [1], did not receive much attention until 1992, when crystal X-ray structure analysis of the enzyme from Saccharomyces cerevisiae was performed [2]. These data, together with the results of site-directed mutagenesis, made it possible to understand in detail the mechanism of thiamine diphosphate-dependent catalysis. Some progress was also made in studies of the functional properties of transketolase. The last review on transketolase, which was fairly complete, appeared in 1998 [3]. Therefore, the publication of this paper should not seem premature.     

Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.Abbreviations PDH pyruvate dehydrogenase complex (EC 1.2.2.2) - PFL pyruvate formate lyase (EC 2.3.1.54) - LDH lactate dehydrogenase (EC 1.1.1.27) - FBP fructose-1,6-bisphosphate - MTT 3-(4,5-dimethyl-thiazoyl-2)-2,5-diphenyltetrazolium bromide - TPP thiamine pyrophosphate     

The affinity chromatography of transketolase.

A number of possible affinity adsorbents for transketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphateglycolaldehydetransferase, EC 2.2.1.1) were prepared. The behaviour of the enzyme from Candida utilis and from Baker's yeast on columns of these and of Blue Sepharose CL-6B was examined, together with the behaviour of the contaminating enzyme, ribulose 5-phosphate 3-epimerase (EC 5.1.3.1). A procedure for removing bound thiamine pyrophosphate by dialysis against EDTA was developed. The competitive inhibition of transketolase by oxythiamine and neopyrithiamine was measured and the Ki values obtained of 1.4 and 4.3 mM, respectively, were compared with the affinity of adsorbents prepared from these two inhibitors. Adsorbents containing bound thiamine pyrophosphate were relatively ineffective but those containing epoxy-linked neopyrithiamine and D-ribose 5-phosphate adsorbed the enzyme at pH 7.4 and it could be eluted in a specific manner.     

Effects of maternal thiamine deficiency on the development of thiamine-dependent enzymes in regions of the rat brain

Previous studies suggest that developing rat brain is susceptible to reduced thiamine intake. In order to assess the metabolic basis for this susceptibility, activities of three thiamine-dependent enzymes (pyruvate dehydrogenase complex, -ketoglutarate dehydrogenase and transketolase) were measured in homogenates of brain tissue from the offspring of thiamine-deficient mothers. Control groups of animals were pair-fed to equal food consumption with the thiamine-deficient animals. The study revealed region-selective delays in the establishment of adult activities of thiamine-dependent enzymes as a result of maternal thiamine deficiency. Pyruvate dehydrogenase complex activities in cerebral cortex were significantly reduced (by 20% P < 0.05); -ketoglutarate dehydrogenase activities were also reduced in cerebral cortex (by 30% P < 0.05). In the case of transketolase, enzyme activities were significantly reduced in cerebral cortex, cerebellum and brainstem. Following thiamine replenishment, defective enzyme activities were restored to normal in all cases. However, since thiamine-dependent enzymes are important for the establishment of adult patterns of cerebral energy metabolism and also in myelin synthesis, maternal thiamine deficiency resulting in reductions of thiamine-dependent enzymes at a vulnerable period in brain development could have serious metabolic consequences leading to permanent neurological sequellae in the offspring.     

Metabolism of transketolase coenzyme in the rat liver

Kinetic analysis permitted to determine two sites of hydroxythiamine diphosphate binding in apotransketolase. The Ki values for these sites differed significantly: (7-22) X 10(-9) M and (13.0-19.7) X 10(-8) M. The rate of thiamine diphosphate turnover within holotransketolase in rat liver tissue was studied by the radioisotope method, using [14C]thiamine as a labeled precursor. The absolute values of half-substitution time and the rate constant of coenzyme degradation in the transketolase molecule are close to those for the protein moiety of the enzyme and are 153 hours and 0.108 days-1, respectively. In vivo rat liver transketolase exists in a substituted alpha-carbanion form. Within the holoenzyme molecule substitution of thiamine diphosphate for hydroxythiamine diphosphate does not influence the formation of an intermediate alpha-carbanion form of the enzyme.     

Half-of-the-site reactivity of the decarboxylating component of the pyruvate dehydrogenase complex from pigeon breast muscle with respect to 2-hydroxyethyl thiamine pyrophosphate

The holopyruvate dehydrogenase is characterized by the charge transfer complex formation between tryptophan residue and thiamine pyrophosphate in each of two active centres. Interaction of apoenzyme with one mole of 2-hydroxyethyl thiamine pyrophosphate results in appearance of the same spectral band which does not change in intensity with further increase in ligand concentration. 2-hydroxyethyl thiamine pyrophosphate: acceptor oxidoreductase activity abolishes after oxidation of only one tryptophan residue per mole of the protein or blocking of one of the active centres with inactive analogue of the coenzyme. In the latter case the charge transfer complex band induced by interaction of apoenzyme with 2-hydroxyethyl thiamine pyrophosphate was not shown at all. These facts testify to half-of-the-site reactivity of pyruvate dehydrogenase with respect to 2-hydroxyethyl thiamine pyrophosphate.     

Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties.

Procedures are described for isolating highly purified porcine liver pyruvate and α-ketoglutarate dehydrogenase complexes. Rabbit serum stabilized these enzyme complexes in mitochondrial extracts, apparently by inhibiting lysosomal proteases. The complexes were purified by a three-step procedure involving fractionation with polyethylene glycol, pelleting through 12.5% sucrose, and a second fractionation under altered conditions with polyethylene glycol. Sedimentation equilibrium studies gave a molecular weight of 7.2 × 10⁶ for the liver pyruvate dehydrogenase complex. Kinetic parameters are presented for the reaction catalyzed by the pyruvate dehydrogenase complex and for the regulatory reactions catalyzed by the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. For the overall catalytic reaction, the competitive K_i to K_m ratio for NADH versus NAD⁺ and acetyl CoA versus CoA were 4.7 and 5.2, respectively. Near maximal stimulations of pyruvate dehydrogenase kinase by NADH and acetyl CoA were observed at NADH:NAD⁺ and acetyl CoA:CoA ratios of 0.15 and 0.5, respectively. The much lower ratios required for enhanced inactivation of the complex by pyruvate dehydrogenase kinase than for product inhibition indicate that the level of activity of the regulatory enzyme is not directly determined by the relative affinity of substrates and products of catalytic sites in the pyruvate dehydrogenase complex. In the pyruvate dehydrogenase kinase reaction, K⁺ and NH⁺₄ decreased the K_m for ATP and the competitive inhibition constants for ADP and (β,γ-methylene)adenosine triphosphate. Thiamine pyrophosphate strongly inhibited kinase activity. A high concentration of ADP did not alter the degree of inhibition by thiamine pyrophosphate nor did it increase the concentration of thiamine pyrophosphate required for half-maximal inhibition.     

Crystal versus solution structures of thiamine diphosphate-dependent enzymes

The quaternary structures of the thiamine diphosphate-dependent enzymes transketolase (EC 2.2.1.1; from Saccharomyces cerevisiae), pyruvate oxidase (EC 1.2.3.3; from Lactobacillus plantarum), and pyruvate decarboxylase (EC 4.1.1.1; from Zymomonas mobilis and brewers' yeast, the latter in the native and pyruvamide-activated forms) were examined by synchrotron x-ray solution scattering. The experimental scattering data were compared with the curves calculated from the crystallographic models of these multisubunit enzymes. For all enzymes noted above, except the very compact pyruvate decarboxylase from Z. mobilis, there were significant differences between the experimental and calculated profiles. The changes in relative positions of the subunits in solution were determined by rigid body refinement. For pyruvate oxidase and transketolase, which have tight intersubunit contacts in the crystal, relatively small modifications of the quaternary structure (root mean square displacements of 0.23 and 0.27 nm, respectively) sufficed to fit the experimental data. For the enzymes with looser contacts (the native and activated forms of yeast pyruvate decarboxylase), large modifications of the crystallographic models (root mean square displacements of 0.58 and 1.53 nm, respectively) were required. A clear correlation was observed between the magnitude of the distortions induced by the crystal environment and the interfacial area between subunits.     

Thiamine pyrophosphokinase deficiency in encephalopathic children with defects in the pyruvate oxidation pathway

Thiamine pyrophosphate (TPP) is an essential cofactor of the cytosolic transketolase and of three mitochondrial enzymes involved in the oxidative decarboxylation of either pyruvate, α-ketoglutarate or branched chain amino acids. Thiamine is taken up by specific transporters into the cell and converted to the active TPP by thiamine pyrophosphokinase (TPK) in the cytosol from where it can be transported into mitochondria. Here, we report five individuals from three families presenting with variable degrees of ataxia, psychomotor retardation, progressive dystonia, and lactic acidosis. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate but normal pyruvate dehydrogenase complex activity in the presence of excess TPP. A reduced concentration of TPP was found in the muscle and blood. Mutation analysis of TPK1 uncovered three missense, one splice-site, and one frameshift mutation resulting in decreased TPK protein levels.     

Regulation of malate oxidation in isolated mung bean mitochondria: I. Effects of oxaloacetate, pyruvate, and thiamine pyrophosphate

In order to investigate the relationship between malate oxidation and subsequent cycle reactions, the effects of oxaloacetate, pyruvate, and thiamine pyrophosphate on malate oxidation in mung bean (Phaseolus aureus var. Jumbo) hypocotyl mitochondria were quantitatively examined. Malate oxidation was optimally stimulated by addition of pyruvate and thiamine pyrophosphate, whose addition lowered the apparent Km for malate from 5 mm to 0.1 mm. Intermediate analysis showed that the stimulatory effect was correlated with removal of oxaloacetate to citrate. Oxaloacetate added alone was shown not to be metabolized until addition of pyruvate and thiamine pyrophosphate; then oxaloacetate was converted in part to pyruvate and also to citrate. These results establish that malate oxidation in mung bean mitochondria is subject to control by oxaloacetate levels, which are primarily determined by the resultant of the activities of malate dehydrogenase, citrate synthase, and pyruvate dehydrogenase.     

Crystal structure of 1-deoxy-D-xylulose 5-phosphate synthase, a crucial enzyme for isoprenoids biosynthesis

Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from Escherichia coli and Deinococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C-2 atom of its thiazolium ring is exposed to a pocket that is the substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.