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PubMed1987

Thiamin uptake in Euglena gracilis.

Thiamin uptake has been investigated in Euglena gracilis Z. This protozoon possessed an active transport system for thiamin with a Km value of 17 nM and a Vmax value of 7.8 pmol per 10(6) cells per min. Thiamin uptake was dependent on pH and temperature, but not on exogenous glucose as an energy source. Oxythiamin and pyrithiamin were competitive inhibitors with Ki values of 33 nM and 15 nM, respectively. Thiamin monophosphate, thiamin pyrophosphate, thiamin triphosphate, heteropyrithiamin, quinolinothiamin, thiamin chloride and amprolium inhibited uptake. Inhibition of thiamin uptake by various metabolic inhibitors and anaerobiosis suggest that thiamin uptake requires an energy source generated by respiration and glycolysis.

Anaerobiosis↗
PubMed1976

Transition state analogs for thiamin pyrophosphate-dependent enzymes.

Many of the transition states that are formed from thiamin pyrophosphate in enzymic reactions are expected to have structures in which the thiazolium ring of thiamin pyrophosphate has lost most of its positive charge. We have synthesized thiamin thiazolone pyrophosphate from the unphosphorylated compound. The sulphur-containing ring of thiamin thiazolone pyrophosphate is uncharged, and thus the compound resembles these transition states. In agreement with the prediction from the transition state theory of reaction rates, thiamin thiazolone pyrophosphate binds to Escherichia coli pyruvate dehydrogenase complex (EC 1.2.7.1) much more strongly than thiamin pyrophosphate itself. An upper limit for the value of the dissociation constant, calculated from the extent of inactivation of the enzyme by a low concentration of thiamin thiazolone pyrophosphate, is 5 X 10(-10) M at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. The dissociation constant for thiamin pyrophosphate under similar conditions is about 10(-5) M. The kinetics of inactivation of pyruvate dehydrogenase complex by thiamin thiazolone pyrophosphate are first order with respect to both enzyme and thiamin thiazolone pyrophosphate; the value of the second order rate constant is 5.7 X 10(5) M-1 min-1 at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. An analysis of the decrease in the rates of inactivation caused by thiamin pyrophosphate indicates that thiamin thiazolone pyrophosphate binds at the coenzyme sites. We have also synthesized thiamin thiothiazolone pyrophosphate and obtained very similar results with this compound.

Alkaline Phosphatase↗
PubMed2003

Effect of intravenous infusions of thiamine on the disposition kinetics of thiamine and its pyrophosphate.

BACKGROUND: Thiamine supplementation is necessary in patients with thiamine deficiency syndromes. Experimental evidence suggests that tissue uptake and the elimination of thiamine are dose-dependent. AIM: The aim of the present study was to investigate the effect of different i.v. infusion rates of thiamine on blood concentrations of thiamine and its active metabolite thiamine pyrophosphate (TPP) and on renal excretion of thiamine. METHODS: Twelve healthy subjects received in a two-period block randomized study 150 mg thiamine intravenously over either 1 or 24 h. RESULTS: The maximum blood concentrations (Cmax) of thiamine were significantly higher after the more rapid infusion (RI; 2300 ng/mL) than after the slower infusion (SI; 177 ng/mL). The AUC of thiamine was identical after both infusion protocols. There was a slightly (10%) increased AUC of TPP (P < 0.08) after SI, whereas C(max) values were comparable. Urinary excretion of thiamine was significantly decreased from 83.6% of the applied dose after RI to 57.6% after the SI. CONCLUSIONS: Our data suggest an increased tissue uptake of thiamine when it is given as an SI compared with a RI of the same dose. It is concluded, therefore, that an SI of thiamine may be superior to RI or bolus injections to treat severe deficiency syndromes.

Adult↗
PubMed1996

Thiamine pyrophosphate-requiring enzymes are altered during pyrithiamine-induced thiamine deficiency in cultured human lymphoblasts.

The most common of the severe complications of thiamine deficiency are beriberi and Wernicke-Korsakoff syndrome. To help clarify the biochemical basis for these disorders, a cell culture system has been established in which pyrithiamine, a potent thiamine transport inhibitor, was used to mimic different degrees of thiamine deficiency within human lymphoblasts. Activities of both transketolase and alpha-ketoglutarate dehydrogenase (alpha-KGDH) decreased at the same rate and to roughly the same levels in response to thiamine deficiency within a given cell line. However, variation in sensitivity to thiamine deficiency, as judged by the relative percentage of loss of enzymatic activities, was found when different cell lines were compared. When exogenous thiamine pyrophosphate was added to the activity assays, differences between transketolase and alpha-KGDH became readily apparent. Only 25% of the lost transketolase activity was present as apo-enzyme, whereas 70% of the lost alpha-KGDH activity was present in the apo-enzyme form. For transketolase, the non-recoverable activity was due mainly to a decrease in the synthesis rate of the protein during thiamine deficiency, suggesting that thiamine has a direct effect on the expression of the transketolase gene and/or protein.

Cell Culture Techniques↗
PubMed1980

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 Ki 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.

Acetaldehyde↗
PubMed1975

Effects of betel nut and fermented fish on the thiamin status of northeastern Thais.

Thiamin deficiency could result either from inadequate intake of thiamin or consumption of food containing antithiamin factors. Dietary surveys conducted in northeastern Thailand indicated that both thiamin and caloric intake of subjects under studies were sufficient. In Thailand, correlation exists between the consumption of food containing antithiamin factors and the prevalence of thiamin deficiency. Betel nuts and raw fermented fish possess antithiamin activity. Abstention from both betel nut chewing and raw fermented fish consumption resulted in a significant reduction of thiamin pyrophosphate effect. The thiamin pyrophosphate effect again increased significantly when the subjects resume their chewing habits. Cooking of fermented fish destroyed thiaminase, resulted in a significant decrease of thiamin pyrophosphate effect of the subjects. Thiamin supplementation (10 mg/day) could further reduce their thiamin pyrophosphate effect. This amount of thiamin could counteract the effect of raw fermented fish consumption but was not sufficient to neutralize the effect of betel nut chewing.

Adolescent↗
PubMed2003

Structural basis for flip-flop action of thiamin pyrophosphate-dependent enzymes revealed by human pyruvate dehydrogenase.

The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the Calpha-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-A resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-A shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.

Amino Acid Sequence↗
PubMed1983

Determination of thiamin and thiamin phosphate esters in blood by liquid chromatography with post-column derivatization.

We demonstrate a liquid-chromatographic method involving post-column derivatization for determining the concentration of thiamin and its phosphate esters in human blood. Blood, erythrocytes, or plasma is deproteinized and centrifuged. Aliquots of the samples are applied to a mu Bondapak C18 column attached to a "high-performance" liquid chromatograph. Addition of potassium ferricyanide/sodium hydroxide solution to the column effluent with a proportioning pump converts thiamin phosphates into fluorophores, the intensities of which are measured with a spectrofluorophotometer. Thiamin, thiamin monophosphate, thiamin pyrophosphate, and thiamin triphosphate eluted as single peaks; no coeluting substances were detected. Thiamin pyrophosphate was the ester present in greatest concentration, followed by thiamin triphosphate; thiamin monophosphate and thiamin were present in slight amounts. This method allows easy determination of thiamin and its phosphate esters in 0.1 mL of blood.

Chromatography, High Pressure Liquid↗
PubMed1990

Bovine heart pyruvate dehydrogenase kinase stimulation by alpha-ketoisovalerate.

Purified bovine heart pyruvate dehydrogenase complex was used to investigate the effects of monovalent cations and alpha-ketoisovalerate on pyruvate dehydrogenase (PDH) kinase inhibition by thiamin pyrophosphate. Initial velocity patterns for thiamin pyrophosphate inhibition were consistent with hyperbolic non-competitive or hyperbolic uncompetitive inhibition at various K+ concentrations between 0 and 120 mM. The Kis, Kid, and Kin for thiamin pyrophosphate were in the range of 0.009 to 5.1 microM over the range of K+ concentrations tested. In the absence of K+, 1 mM alpha-ketoisovalerate had no effect on PDH kinase inhibition by thiamin pyrophosphate, whereas in the presence of 20 mM K+, alpha-ketoisovalerate stimulated PDH kinase activity almost 2-fold over the range of 0-80 microM thiamin pyrophosphate. Half-maximal stimulation by alpha-ketoisovalerate occurred at about 200 microM in the presence of 100 microM thiamin pyrophosphate and 20 mM K+. Similar but less extensive changes occurred in the presence of 100 microM thiamin pyrophosphate and 1 mM NH4+. Initial velocity patterns for PDH kinase inhibition by thiamin pyrophosphate in the presence of 2 mM alpha-ketoisovalerate were mixed noncompetitive, but alpha-ketoisovalerate increased the Vm and Km for adenosine 5'-triphosphate in the presence of inhibitor. In the presence of thiamin pyrophosphate, PDH kinase remained stimulated after chromatography on Sephadex G-25 to remove alpha-ketoisovalerate. The results indicate that acylation of pyruvate dehydrogenase complex by alpha-ketoisovalerate results in PDH kinase stimulation but only in the presence of monovalent cations and thiamin pyrophosphate.

Acylation↗
PubMed1983

Quantitative analysis of total thiamine in human blood, milk and cerebrospinal fluid by reversed-phase ion-pair high-performance liquid chromatography.

Whole blood hemolysed by freezing, serum, cerebrospinal fluid, and milk of human origin were deproteinized by perchloric acid. Thiamine pyrophosphate and thiamine monophosphate were hydrolysed to thiamine by acid phosphatase. Chromatography was performed on C18-coated silica using an n-octanesulfonate containing mobile phase methanol-aqueous citrate buffer pH 4.0 (45:55, v/v). In a post-column reaction K3Fe(CN)6 is used to oxidise thiamine to thiochrome, which is detected by fluorometry. Two ml blood is needed. The minimum detectable amount is 60 femtomol of thiamine. The intra-assay coefficient of variation (C.V.) is 2.3% and the inter-assay C.V. is 3.9%. The recovery of added thiamine pyrophosphate to blood samples was 98.7%. The reference range was found to be 88-157 nmol/1 whole blood. Examples of the analysis of cerebrospinal fluid, serum and milk are given.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

[Interrelation between the content of thiamine phosphates and pyruvate dehydrogenase activity in the liver of rats with intensified lipogenesis].

Injection of thiamine (250 micrograms per 100 g of mass daily) to rats with alimentary intensification of lipogenesis decreases the pyruvate dehydrogenase activity in the liver, which is accompanied by an increase in the concentration of pyruvate and drop in the level of free fatty acids. Under these conditions the content of total thiamine is 1.5 times as high. The level of thiamine pyrophosphate and thiamine triphosphate rises significantly. When all parameters were determined in dynamics after a single thiamine injection, an inverse dependence is observed between the content of thiamine phosphoric esters, thiamine triphosphate in particular, and pyruvate dehydrogenase activity.

Animals↗
PubMed1989

2-Acetylthiamin pyrophosphate: an enzyme-bound intermediate in thiamin pyrophosphate-dependent reactions.

Several enzymes catalyze reactions that may involve acetylthiamin pyrophosphate (acetyl-TPP) as an intermediate. These enzymes are phosphoketolase, pyruvate oxidase and several pyruvate oxidoreductases. Acetyl-TPP can be synthesized and used as a carrier to analyze quenched reaction mixtures for the presence of [14C]acetyl-TPP. Synthetic acetyl-TPP exhibits unusual chemical properties and a unique pH-rate profile that serves as a powerful means of characterizing [14C]acetyl-TPP that has been isolated from quenched enzymatic reaction mixtures. Using this and other methods, extensive evidence has been obtained for the involvement of acetyl-TPP in certain reactions catalyzed by the pyruvate dehydrogenase complex (PDH complex) of Escherichia coli. Acetyl-TPP is chemically competent as an intermediate in the decarboxylation and dehydrogenation of pyruvate by the PDH complex; and it is transiently formed during the course of this reaction. It may be an enzyme-bound intermediate or it may be in equilibrium with such an intermediate. Acetyl-TPP is very likely to be an intermediate of the phosphoketolase reaction. However, no direct evidence linking it to the phosphoketolase reaction mechanism is yet available. It is unclear whether acetyl-TPP is an intermediate in the pyruvate oxidoreductase reactions. In one example, that of the ketoacid oxidoreductase of Halobacterium halobium, analysis by electron paramagnetic resonance spectroscopy indicates the involvement of a hydroxyethyl-TPP-radical as an intermediate. It is unknown whether the subsequent reaction of this radical with coenzyme A an an oxidized FeS cluster to produce acetyl coenzyme A and the reduced cluster involves the intermediate formation of acetyl-TPP.

Aldehyde-Lyases↗
PubMed1983

The catalytic mechanism of transketolase. Thiamin pyrophosphate-derived transition states for transketolase and pyruvate dehydrogenase are not identical.

Thiamin thiazolone pyrophosphate (TTPP) has been reported to be an effective transition state analogue for the thiamin pyrophosphate-dependent partial reaction of pyruvate dehydrogenase (Gutowski, J. A., and Lienhard, G. E. (1976) J. Biol. Chem. 251, 2863-2866). The kinetics of the interaction of TTPP with transketolase are reported here. TTPP is a competitive inhibitor, with respect to thiamin pyrophosphate, of bakers' yeast transketolase but it is neither a tight binding inhibitor nor a slow binding inhibitor. TTPP decreases the kinetically observed negative cooperativity seen for thiamin pyrophosphate and also decreases the rate constant for the hysteretic activation of the enzyme by thiamin pyrophosphate. We conclude that thiamin thiazolone pyrophosphate is not an effective transition state analogue for the reaction catalyzed by bakers' yeast transketolase. This difference between transketolase and pyruvate dehydrogenase may be related to differences in the polarity of the active sites of the enzymes. It is conceivable that the active sites of the pyruvate decarboxylase subunit of pyruvate dehydrogenase is hydrophobic, by analogy with the known hydrophobicity of the active site of brewers' yeast pyruvate decarboxylase. This hydrophobicity would stabilize a transition state with no charge on the thiazole portion of the coenzyme, similar to the "uncharged" thiazole portion of TTPP. In contrast, the active site of bakers' yeast transketolase, which is known to contain charged amino acid side chains, should be less favorable for such an uncharged transition state. A charge-separated canonical form related to TTPP could be preferentially stabilized in the active site of transketolase.

Kinetics↗
PubMed2004

Obese individuals as thiamin storers.

OBJECTIVE: To investigate thiamin and its phosphoester content in plasma and erythrocytes for a complete picture of thiamin status in obese individuals. DESIGN: Comparative study of the thiamin status of obese vs normal individuals. SUBJECTS: In all, 10 healthy, overweight, fertile age women (age: 33.1+/-5.1 y; BMI: 47.0+/-0.2 kg/m(2)) and 10 normal women (age: 30.1+/-3.5 y; BMI: 22.8+/-0.2 kg/m(2)). METHODS: a high-pressure liquid chromatography (HPLC) method for the determination of thiamin and its phosphoesters in the plasma and erythrocytes of the subjects. RESULTS: The major findings were: (1) significant decrease of plasma thiamin, its monophosphate and total thiamin contents in obese vs normal women; (2) significant decrease of thiamin pyrophosphate ester and total thiamin content in obese vs normal women; (3) significant increase in plasma thiamin/thiamin monophosphate ratio (in practice, it was inverted) and corresponding decrease of the plasma thiamin monophosphate/erythrocytes thiamin pyrophosphate ratio in obese vs normal women, where plasma thiamin monophosphate and erythrocytes thiamin pyrophosphate contents are an index of thiamin status. CONCLUSIONS: This study advances the hypothesis that obese women maintain higher levels of thiamin compared to normal weight subjects by storing greater amounts of thiamin in cells through preferential intracellular thiamin recycling to compensate for relatively lower levels of thiamin.

Adult↗
PubMed2001

The mechanism of action of bacimethrin, a naturally occurring thiamin antimetabolite.

The mechanism of bacimethrin (2) toxicity has been determined. This compound is converted to 2'-methoxy-thiamin pyrophosphate (10) by the thiamin biosynthetic enzymes. Of the seven thiamin pyrophosphate utilizing enzymes in Escherichia coli, 2'-methoxy-thiamin pyrophosphate inhibits alpha-ketoglutarate dehydrogenase, transketolase, and deoxy-D-xylulose-5-phosphate synthase. Bacimethrin does not cause repression of the genes coding for the thiamin biosynthetic enzymes.

Antimetabolites↗
PubMed1974

Regulation of heart muscle pyruvate dehydrogenase kinase.

1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [(32)P]phosphate from [gamma-(32)P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100mum) and cyclic 3':5'-nucleotides (at 10mum) had no significant effect on kinase activity. 3. The K(m) for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76mum. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The K(m) for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9-25.4mum. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The K(m) for pyruvate in the pyruvate dehydrogenase reaction was 35.5mum. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25-500mum. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10mum) the kinase activity was enhanced by low concentrations of pyruvate (25-100mum) and inhibited by a high concentration (500mum). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms (14)CO(2) from [1-(14)C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg(2+) (15mm) and by Ca(2+) (10nm-10mum) at low Mg(2+) (0.15mm) but not at high Mg(2+) (15mm).

Animals↗