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PubMed1984

Localization of tryptophan residues in thiamine pyrophosphate-binding sites of pyruvate dehydrogenase from pigeon breast muscle.

Interaction of N-bromosuccinimide with the pyruvate dehydrogenase component of the pigeon breast muscle pyruvate dehydrogenase complex results in a rapid and specific modification of two tryptophan residues per mole of the protein and in complete inactivation of the enzyme. Modification of the enzyme excludes the development of the negative Cotton effect with a maximum at 330 nm, characteristic of the charge transfer complex between the protein tryptophan residue and thiamine pyrophosphate. Modification of one tryptophan residue was shown to result in the absence of the band at 330 nm in one of the active centers of pyruvate dehydrogenase, while oxidation of two tryptophan residues excludes the formation of a charge transfer complex in both centers. The conclusion was drawn about the presence in the pyruvate dehydrogenase active centers of two tryptophan residues involved in the formation of the charge transfer complex with the thiazolium ring of thiamine pyrophosphate.

Animals↗

PubMed1984

Turnover of [14C]thiamin and activities of thiamin pyrophosphate-dependent enzymes in tissues of mice with Ehrlich ascites carcinoma.

Turnover of [14C]thiamin was studied in mice with Ehrlich ascites carcinoma fed a thiamin-deficient diet and injected with 5 or 2 micrograms/mouse of the labeled vitamin. The process of conversion of [14C]thiamin to thiamin pyrophosphate (TPP) was monitored by measuring the activities of transketolase, pyruvate dehydrogenase, and oxoglutarate dehydrogenase. The amount of coenzyme-unsaturated apotransketolase was assessed by measuring the TPP effect--determining transketolase activity with and without the addition of TPP in vitro. Tumor growth was accompanied by thiamin deficiency, manifested in an increase in [14C]thiamin incorporation into the host tissues and the absence of saturation of the tissues with the labeled vitamin over 13 days. Increased values for the turnover coefficients, reduction of thiamin-dependent enzyme activities, elevation of the TPP effect, and a decrease in urinary excretion of the radioactive products also provided evidence for a disturbance in thiamin metabolism. The severity of the disturbance in thiamin metabolism during malignant tumor growth was directly related to the dose of the exogenous vitamin.

Animals↗

PubMed1989

A common structural motif in thiamin pyrophosphate-binding enzymes.

The amino acid sequences of a wide range of enzymes that utilize thiamin pyrophosphate (TPP) as cofactor have been compared. A common sequence motif approximately 30 residues in length was detected, beginning with the highly conserved sequence -GDG- and concluding with the highly conserved sequence -NN-. Secondary structure predictions suggest that the motif may adopt a beta alpha beta fold. The same motif was recognised in the primary structure of a protein deduced from the DNA sequence of a hitherto unassigned open reading frame of Rhodobacter capsulata. This putative protein exhibits additional homology with some but not all of the TPP-binding enzymes.

Amino Acid Sequence↗

PubMed1984

Escherichia coli pyruvate dehydrogenase complex. Thiamin pyrophosphate-dependent inactivation by 3-bromopyruvate.

Inactivation of the pyruvate dehydrogenase complex by 3-bromopyruvate is thiamin pyrophosphate (TPP)-dependent. Inactivation with 2-14C- or 3-14C-labeled 3-bromopyruvate results in TPP-dependent covalent labeling of more than 60 sites in the complex, all of which are associated with the dihydrolipoyl transacetylase component. Inactivation by 3-bromo[1-14C]pyruvate labels up to 20 sites associated with dihydrolipoyl transacetylase, also with TPP dependence. Systemic chemical degradation of the complex inactivated by 3-bromo[2-14C]pyruvate under conditions that would convert lipoyl groups to S,S,-biscarboxymethyl dihydrolipoic acid produces S,S,-bis[14C]carboxymethyl dihydrolipoic acid. It is concluded that 3-bromopyruvate inactivates this complex by initially undergoing the first two steps of the usual catalytic pathway, TPP-dependent decarboxylation followed by reductive bromoacetylation of lipoyl moieties. The sulfhydryl groups of S-bromoacetyl dihydrolipoyl moieties generated by reductive bromoacetylation are then alkylated by 3-bromopyruvate as well as by bromoacetyl thioester groups associated with the complex.

Affinity Labels↗

PubMed1975

Leigh's disease: significance of the biochemical changes in brain.

Analysis of five brains from patients with Leigh's disease demonstrates an accumulation of thiamine pyrophosphate and a deficiency of thiamine triphosphate. The enzyme which converts thiamine pyrophosphate to thiamine triphosphate was normally active in two of these brains, suggesting that the inhibitor found in Leigh's disease is probably producing the observed neurochemical changes. Reasons for the histological similarity between Leigh's and Wernicke's diseases are suggested.

Brain↗

PubMed2005

Genetic regulation mediated by thiamin pyrophosphate-binding motif in Saccharomyces cerevisiae.

The expression of genes of Saccharomyces cerevisiae encoding the enzymes involved in the metabolism of thiamin (THI genes) is co-ordinately repressed by exogenous thiamin and induced in the absence of thiamin. In this yeast THI regulatory system acts mainly at the transcriptional level, thiamin pyrophosphate (TDP) seems to serve as a corepressor, and genetic studies have identified three positive regulatory factors (Thi2p, Thi3p and Pdc2p). We found in a DNA microarray analysis that the expression of THI genes increased 10- to 90-fold in response to thiamin deprivation, and likewise, the expression of THI2 and THI3 increased 17-fold and threefold, respectively. After transfer from repressing to inducing medium, the promoter activity of both THI2 and THI3 increased in parallel with that of PHO3, one of THI genes. The stimulation of THI3 promoter activity was diminished by deletion of THI3, indicative of the autoregulation of THI3. The THI genes were not induced when THI2 was expressed from the yeast GAL1 promoter in a thi3Delta strain or when THI3 was expressed in a thi2Delta strain, suggesting that Thi2p and Thi3p participate simultaneously in the induction. When mutant Thi3p proteins lacking TDP-binding activity were produced in the thi3Delta strain, THI genes were expressed even under thiamin-replete conditions. This result supports the hypothesis that Thi3p senses the intracellular signal of the THI regulatory system to exert transcriptional control. Furthermore, Thi2p and Thi3p were demonstrated to bind each other and this interaction was partially diminished by exogenous thiamin, suggesting that Thi2p and Thi3p stimulate the expression as a complex whose function is disturbed by TDP bound to Thi3p. We discuss the possibility that the induction of THI genes is triggered by the activation of the complex attributed to decrease in intracellular TDP and the elevated complex in the autoregulatory fashion further upregulates THI genes. This is the first report of the involvement of the TDP-binding motif in genetic regulation.

Cell Nucleus↗

PubMed1999

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during alpha-oxidation of 3-methyl-branched fatty acids.

In the third step of the alpha-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde. The cleavage enzyme was purified from the matrix protein fraction of rat liver peroxisomes and identified as a protein made up of four identical subunits of 63 kDa. Its activity proved to depend on Mg(2+) and thiamine pyrophosphate, a hitherto unrecognized cofactor of alpha-oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction products when the purified enzyme was incubated with 2-hydroxy-3-methylhexadecanoyl-CoA as the substrate. Hence the enzyme catalyzes a carbon-carbon cleavage, and we propose calling it 2-hydroxyphytanoyl-CoA lyase. Sequences derived from tryptic peptides of the purified rat protein were used as queries to recover human expressed sequence tags from the databases. The composite cDNA sequence of the human lyase contained an ORF of 1,734 bases that encodes a polypeptide with a calculated molecular mass of 63,732 Da. Recombinant human protein, expressed in mammalian cells, exhibited lyase activity. The lyase displayed homology to a putative Caenorhabditis elegans protein that resembles bacterial oxalyl-CoA decarboxylases. Similarly to the decarboxylases, a thiamine pyrophosphate-binding consensus domain was present in the C-terminal part of the lyase. Although no peroxisome targeting signal, neither 1 nor 2, was apparent, transfection experiments with constructs encoding green fluorescent protein fused to the full-length lyase or its C-terminal pentapeptide indicated that the C terminus of the lyase represents a peroxisome targeting signal 1 variant.

Amino Acid Sequence↗

PubMed2000

Is thiamine deficiency in elderly people related to age or co-morbidity?

OBJECTIVES: to compare erythrocyte thiamine pyrophosphate concentrations in elderly people with those in healthy younger people; to determine if any differences can be attributed to age or to co-morbidities. DESIGN: cross-sectional and 3-year longitudinal surveys. SETTING: primary care. PATIENTS: 100 volunteer blood donors and 222 elderly people from a general practice register. MEASUREMENTS: thiamine pyrophosphate concentrations using high performance liquid chromatography; physical examination, medical and medication history; grip strength, body mass index and plasma albumin. RESULTS: the mean [95% confidence interval (CI)] thiamine pyrophosphate concentration was 152 nmol/l (147-158) in the elderly group and 224 (213-235) nmol/l in the younger group (P < 0.001). Ninety-six (43.4%) of the elderly subjects had thiamine pyrophosphate concentrations below the fifth percentile of the younger subjects (140 nmol/l). Over 3 years thiamine pyrophosphate concentrations fell in the elderly cohort by 20% (95% CI: 14.5-24.5%; P < 0.01). Thiamine pyrophosphate concentrations in 39 healthy older people were no different from those in elderly people with co-morbidity but were significantly lower than those in the younger people. Elderly people with absent vibration sense in their feet had a lower thiamine pyrophosphate concentration than the rest of the group [129 (117-142)nmol/l compared with 156 (150-162)nmol/l; P < 0.01)]. Thiamine pyrophosphate concentrations were not related to prevalent diseases, common medications, body mass index, grip strength or plasma albumin. CONCLUSION: lower thiamine pyrophosphate concentrations in elderly people appear to be related more to age itself than to co-existent illnesses.

Adult↗

PubMed1984

Effect of oxythiamine on adrenal thiamine pyrophosphate-dependent enzyme activities.

The effect of parenterally administered low doses of oxythiamine (0.5 mumol/100 g body weight every 12 h) on rat adrenal transketolase, pyruvate dehydrogenase ( PDC ) and 2-oxoglutarate dehydrogenase complex ( OGDC ) activities was studied. It was found that the transketolase and the PDC activities decreased after 12, 16, and 20 injections. When the activities were measured in the absence of exogenous thiamine pyrophosphate (TPP), the PDC inhibition was significant after 20 oxythiamine injections. The OGDC activity was resistant to oxythiamine administration. The oxythiamine effects seem to be due to its being phosphorylated to yield diphosphate ester which, as kinetic studies on highly purified PDC from bovine adrenals have shown, is a strong competitive inhibitor (Ki = 0.07 microM) with respect to TPP (Km = 0.11 microM). The data obtained provide evidence for the possibility of a partial selective in vivo inhibition of the adrenal TPP-dependent enzymes.

Adrenal Glands↗

PubMed2006

Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch.

Riboswitches are metabolite-sensing RNAs, typically located in the non-coding portions of messenger RNAs, that control the synthesis of metabolite-related proteins. Here we describe a 2.05 angstroms crystal structure of a riboswitch domain from the Escherichia coli thiM mRNA that responds to the coenzyme thiamine pyrophosphate (TPP). TPP is an active form of vitamin B1, an essential participant in many protein-catalysed reactions. Organisms from all three domains of life, including bacteria, plants and fungi, use TPP-sensing riboswitches to control genes responsible for importing or synthesizing thiamine and its phosphorylated derivatives, making this riboswitch class the most widely distributed member of the metabolite-sensing RNA regulatory system. The structure reveals a complex folded RNA in which one subdomain forms an intercalation pocket for the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety of TPP, whereas another subdomain forms a wider pocket that uses bivalent metal ions and water molecules to make bridging contacts to the pyrophosphate moiety of the ligand. The two pockets are positioned to function as a molecular measuring device that recognizes TPP in an extended conformation. The central thiazole moiety is not recognized by the RNA, which explains why the antimicrobial compound pyrithiamine pyrophosphate targets this riboswitch and downregulates the expression of thiamine metabolic genes. Both the natural ligand and its drug-like analogue stabilize secondary and tertiary structure elements that are harnessed by the riboswitch to modulate the synthesis of the proteins coded by the mRNA. In addition, this structure provides insight into how folded RNAs can form precision binding pockets that rival those formed by protein genetic factors.

Base Sequence↗

PubMed1975

Purification and properties of sulfoacetaldehyde sulfo-lyase, a thiamine pyrophosphate-dependent enzyme forming sulfite and acetate.

Sulfoacetaldehyde sulfo-lyase, which decomposes sulfoacetaldehyde to sulfite and acetate, was extracted from a bacterium grown on taurine, and purified, and characterized. A method for assay of enzyme activity was devised on formation of a bisulfite adduct with benzaldehyde. The enzyme was purified 14-fold from an extract of cells grown on taurine and appeared homogeneous on disc-electrophoresis. The molecular weight of the enzyme was estimated to be 85,000 by gel filtration. The enzyme required thiamine pyrophosphate (TPP) and Mg2+ for activity and preincubation with TPP and Mg2+ was required for maximum activity. The optimum pH for activity was 7.5. The Km value for TPP was determined to be 2.7 muM and that for sulfoacetaldehyde to be 5.0mM. Sulfite was produced only from sulfoacetaldehyde among a variety of sulfonates tested. rho-Chloromercuribenzoate, EDTA, and sulfite, a reaction product, inhibited the enzyme reaction. The enzyme seemed to be inducible, since activity was found in extracts of cells grown on taurine but not on peptone.

Acetaldehyde↗

PubMed1982

Anorexia and antagonism of thiamin utilization in poultry treated with furazolidone.

Furazolidone (0.4% w/w in the feed, 10 days) reduced the feed intake and growth in 9 week old chickens, and increased the amount of 5-hydroxytryptamine (5-HT) in the brain. The drug also increased the stimulation of transketolase activity by thiamine pyrophosphate (TPP) in lysed blood cells (TPP effect), and the concentrations of pyruvate and lactate in the blood. Experiments with pair-fed birds showed that the reduction in feed intake in furazolidone-treated chickens could account for the reduced growth. The drug also produced anorexia in ducklings and turkey poults. In chickens, the anorectic action of furazolidone was unaffected by methergoline (1 mg/kg, twice daily, I.M), and in ducklings furazolidone did not consistently produce anorexia, although it always inhibited monoamine oxidase (MAO) activity in the brain. These observations suggest that the anorectic action of the drug was not tryptaminergic in nature. The increase in the TPP effect found in preparations from furazolidone-treated chickens was absent in preparations from pair-fed birds on unmedicated feed. Thus the TPP effect could be used as an indicator of the effect of the drug on the thiamine status of chickens. However, the increase in the concentrations of pyruvate and lactate in blood was found both in furazolidone-treated birds and pair-fed birds on unmedicated feed, showing that they were attributable to the reduction in feed intake. The TPP effect in furazolidone-treated chickens, 14-49 +/- 2.33% (n = 10), was sufficient to suggest a mild deficiency of thiamin pyrophosphate. Thiamin, given at a dosage above the requirement of the vitamin for chickens, did not reduce the anorexia or the TPP effect of furazolidone-treated birds, although it stimulated the feed intake and growth of birds on unmedicated feed. It is proposed that furazolidone antagonized the utilization of thiamin, perhaps by inhibiting its phosphorylation. Following the withdrawal of furazolidone, the TPP effect returned to the control value and the rate of growth of the birds increased and matched that the controls. Thus the effect of the drug was reversible. Addition of furazolidone to the feed at a concentration of 0.01% w/w for 28 days did not produce anorexia in chickens or affect the amount of 5-HT in the brain. Thus at this level, it is unlikely that the drug would produce adverse effects in poultry.

Animals↗

PubMed1985

Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate.

The pyruvate dehydrogenase multienzyme complex from bovine kidney and heart is inactivated by treatment with pyridoxal 5'-phosphate and sodium cyanide or sodium borohydride. The site of this inhibition is the pyruvate dehydrogenase (E1) component of the complex. Inactivation of E1 by the pyridoxal phosphate-cyanide treatment was prevented by thiamin pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamin pyrophosphate binding sites per molecule (alpha 2 beta 2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 5-fold. Incorporation of approximately 2.4 equiv of 14CN per mole of E1 tetramer in the presence of pyridoxal phosphate resulted in about a 90% loss of E1 activity. Radioactivity was incorporated predominantly into the E1 alpha subunit. Radioactive N6-pyridoxyllysine was identified in an acid hydrolysate of the E1-pyridoxal phosphate complex that had been reduced with NaB3H4. The data are interpreted to indicate that in the presence of sodium cyanide or sodium borohydride, pyridoxal phosphate reacts with a lysine residue at or near the thiamin pyrophosphate binding site of E1. This binding site is apparently located on the alpha subunit.

Animals↗

PubMed1983

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.

2,6-Dichloroindophenol↗

PubMed1995

The thiamine-dependent hysteretic behavior of human transketolase: implications for thiamine deficiency.

We have investigated the hysteretic properties of human transketolase with emphasis on its dependency on thiamine pyrophosphate concentration. As demonstrated previously, the reaction progress curves revealed a slow transition from an initial low velocity to a faster final steady-state velocity, characterized by the rate constant tau-1. The rate of the transition was dependent on the concentration of the thiamine pyrophosphate cofactor, with progressively longer transition times found as the concentration of thiamine pyrophosphate was decreased. At physiological thiamine pyrophosphate concentrations, the inverse rate constant was in the range of 10 to 20 min for fibroblast-derived transketolase and increased dramatically with only small decreases from these levels of thiamine pyrophosphate. Variation in the lag was found when transketolase from different individuals was examined. Moreover, at low levels of thiamine, the rate of the transition was different between fibroblast- and lymphoblast-derived transketolase. The substantial lag in formation of active holoenzyme and the findings of interindividual variation and cell type variation in the lag period suggest mechanisms for the loss of transketolase activity during thiamine deficiency and may explain, at least in part, the differential sensitivity to deficiency demonstrated by tissues and individuals.

Cells, Cultured↗

PubMed1982

Kinetic studies of mouse brain transketolase.

The activity of transketolase in mouse brain was 5.7 nmol/min/mg protein measured by an enzyme-coupled spectrophotometric assay. The apparent Km for ribose-5-phosphate was 330 microM, for D-xylulose-5-phosphate was 120 microM, and for thiamine pyrophosphate was 7 microM. 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.

Animals↗

PubMed1986

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase.

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.

Animals↗

PubMed1996

Thiamine status in patients receiving long-term home parenteral nutrition.

OBJECTIVES: Clinical thiamine deficiency can occur in patients receiving total parenteral nutrition (TPN) without thiamine supplementation. Because considerable breakdown of thiamine may occur in the presence of bisulfite-containing amino acid solutions, subclinical thiamine deficiency may develop with the use of these solutions, even with appropriate thiamine supplementation. The current American Medical Association-Food and Drug Administration approved injectable multivitamin formula contains 3 mg of thiamine. This study was undertaken to determine whether this quantity of thiamine is sufficient to avoid clinical thiamine deficiency in long-term home TPN patients with negligible oral thiamine absorption and in the presence of bisulfite-containing amino acid solutions. METHODS: Twenty-four long-term home TPN patients with oral caloric intakes below the norm were evaluated. Seventeen patients suffered from short bowel syndrome or radiation enteritis, and another three had draining gastrostomies that precluded all intestinal absorption. The duration of TPN therapy ranged between 1 and 164 months. Thiamine status was assessed by assaying thiamine pyrophosphate, transketolase activity, and blood thiamine levels. RESULTS: All thiamine pyrophosphate and erythrocyte transketolase activity levels were within the normal range. CONCLUSIONS: This study demonstrates that the currently recommended 3 mg of thiamine hydrochloride added to TPN solutions is adequate to maintain normal thiamine status. This should prevent the development of thiamine deficiency even in patients with compromised intestinal thiamine absorption, and in the presence of bisulfite-containing amino acid solutions.

Adult↗