Thiamine pyrophosphate-metal ion complexation: equilibrium and kinetics.
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Binding Sites↗
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Binding of thiamin thiazolone pyrophosphate to mammalian pyruvate dehydrogenase and its effects of kinase and phosphatase activities.
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Animals↗
Thiamine pyrophosphate-catalyzed enzymatic decarboxylation of -oxo acids.
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Alcohols↗
Alpha-oxidation of 3-methyl-substituted fatty acids and its thiamine dependence.
3-Methyl-branched fatty acids, as phytanic acid, undergo peroxisomal alpha-oxidation in which they are shortened by 1 carbon atom. This process includes four steps: activation, 2-hydroxylation, thiamine pyrophosphate dependent cleavage and aldehyde dehydrogenation. The thiamine pyrophosphate dependence of the third step is unique in peroxisomal mammalian enzymology. Human pathology due to a deficient alpha-oxidation is mostly linked to mutations in the gene coding for the second enzyme of the sequence, phytanoyl-CoA hydroxylase.
Amino Acid Sequence↗
Complexation of Cu(2+) by HETPP and the pentapeptide Asp-Asp-Asn-Lys-Ile: a structural model of the active site of thiamin-dependent enzymes in solution.
To obtain structural information on the active site of thiamin-dependent enzymes in solution, we have studied the interactions of Cu(2+) ions with 2-(alpha-hydroxyethyl)thiamin pyrophosphate (HETPP), the pentapeptide Asp-Asp-Asn-Lys-Ile surrounding the thiamin pyrophosphate moiety in the transketolase enzyme, and the tertiary Cu(2+)-pentapeptide-HETPP system in aqueous solutions at various pH values. In the binary Cu(2+)-pentapeptide system around physiological pH, the bonding sites were the terminal NH2 group, the aspartate beta-carboxylates, and a deprotonated peptide nitrogen, while, in the Cu(2+)-HETPP system at the same pH, the Cu(II) was coordinated to the pyrophosphate group and to the pyrimidine N(1') atom. It is found that, in the tertiary system at physiological pH, the peptide bone offers three coordination sites to the metal ion, and the coordination sphere is completed by two additional phosphate oxygens and the nitrogen N(1') of the thiamin coenzyme. The stability constants in the tertiary system are higher than those in the simpler Cu(2+)-HETPP and Cu(2+)-peptide systems. The present data show that the coenzyme adopts the so-called S conformation in solution. The importance of our findings concerning the N(1') coordination and the S conformation in the tertiary system is discussed in conjunction with the role of HETPP as an intermediate of thiamin catalysis.
Binding Sites↗
Thiamine, thiamine phosphates and thiamine metabolizing enzymes in synaptosomes of rat brain.
Thiamine and thiamine mono-, pyro- and triphosphate were found at detectable levels in synaptosomes isolated from whole rat brain. Synaptosomes prepared from whole brain, cerebellum and medulla were also found to contain uridine and inosine mono- and diphosphatases as well as the thiamine pyrophosphate synthetizing and hydrolyzing enzymes, but no thiamine monophosphatase. By isoelectric focusing on thin layer polyacrylamide gel of Triton X-100 homogenates of synaptosomes, thiamine pyrophosphatase activity could be separated into 10 bands with different isoelectric points. The contents of thiamine compounds and enzymes in synaptosomes were generally lower than those found in neuronal cell bodies.
Animals↗
Formation of N-hydroxy-N-arylacetamides from nitroso aromatic compounds by the mammalian pyruvate dehydrogenase complex.
Bovine, human and porcine heart mitochondria and isolated porcine heart pyruvate dehydrogenase complex (PDHC) pyruvate-dependently form N-hydroxy-N-arylacetamides from nitroso aromatic compounds, including carcinogenic 4-biphenyl and 2-fluorenyl derivatives. The PDHC-catalysed formation of N-hydroxyacetanilide (N-OH-AA) from nitrosobenzene (NOB), through a Ping Pong mechanism, is optimum at pH 6.8 and is accelerated by thiamin pyrophosphate, but is inhibited by thiamin thiazolone pyrophosphate and ATP. Km pyruvate in the reaction is independent of pH over the range tested, whereas KmNOB increases at lower pH, owing to ionization of an active-site functional group of pKa 6.3. The enzymic ionization decreases log (Vmax/KmNOB). Isolated pyruvate dehydrogenase (E1), a constitutive enzyme of PDHC, forms N-OH-AA by itself and has comparable kinetic parameters to those of the PDHC-catalysed N-OH-AA formation. The catalytic efficiency of PDHC in the formation of N-hydroxy-N-arylacylamides, due to the steric limitation of the active site of E1, is lowered both by bulky alkyl groups of alpha-oxo acids and by p-substituents (but not an o-substituent) on nitrosobenzenes. These nitroso compounds serve as electrophiles in the reaction in which the reductive acetylation step is rate-limiting. The reaction mechanism and other factors affecting N-hydroxy-N-arylacylamide formation are discussed.
Acetamides↗
Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae.
Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.
Carboxy-Lyases↗
Investigations on the pyruvate decarboxylase catalysed oxidative decarboxylation of 2-oxoacids by 2.6-dichlorophenolindophenol.
Pyruvate decarboxylase, a thiamine pyrophosphate and Mg2+ dependent enzyme, catalyzes normally the simple decarboxylation of its substrate. However, in the presence of suitable hydrogen acceptors, such as dichlorophenolindophenol, it catalyzes an artificial reaction, the oxidative decarboxylation of 2-oxoacids to the corresponding carboxylic acids. As a result of kinetic studies a mechanism is presented in this paper, which describes the synchronous progress of both the oxidative ping-pong-type decarboxylation reaction and the physiological non-oxidative decarboxylation reaction. Moreover, experiments using phenylglyoxylic acids that carry suitable substituents in the 4-position have shown that the electronic influence of the substituents (causing a decrease in the rate constants with increasing electron pressure) is in quantitative agreement for both types of reactions. A common rate limiting transition state preceding the alpha-carbanion intermediate of the enzymic reaction (2-(1-hydroxybenzyl)-thiamine pyrophosphate carbanion) must therefore be assumed for both reactions. Acetaldehyde which acts as noncompetitive inhibitor in the normal enzyme mechanism does not influence the oxidative decarboxylation reaction. 4'-hydroxy-4'-deamino-thiamine pyrophosphate is inactive as coenzyme for both types of enzyme reactions. This confirms again the essential role of the 4'-amino group in the cofactor function.
2,6-Dichloroindophenol↗
Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.
Tetrahydrothiamin pyrophosphate, an analogue of thiamin pyrophosphate (TPP) in which the thiazolium ring has been reduced to a thiazolidine ring, was prepared by borohydride reduction of TPP. It consists of four stereoisomers, comprising two diastereomers each of which is a racemic mixture, generated by the introduction of two chiral centers on the thiazolidine ring. The major and minor diastereomers were separated and inferred to be of the cis and trans configurations, respectively, from a study of the nuclear Overhauser effects in the 1H NMR spectrum of the simpler tetrahydrothiamin. Tetrahydro-TPP behaves as a mixture of potent inhibitors of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex from Escherichia coli. The site of binding is probably the TPP-binding site on E1, and the Kd for each of the four stereoisomers was estimated. The cis isomers of tetrahydro-TPP bind more tightly than does TPP, whereas the trans isomers appear to bind with about the same Kd as TPP. Sodium borohydride caused a rapid inhibition of E1 activity in the presence of TPP, believed to be due to reaction of borohydride with enzyme-bound TPP. The experiments are consistent with an enhancement of the reactivity of the thiazole ring of TPP when bound to the catalytic site of E1, which could be due to polarization of the greater than +N=C bond near a hydrophobic or positively charged region of the protein. A spontaneous reactivation occurred after the initial inhibition by borohydride, attributable to a weakly binding inhibitor, not tetrahydro-TPP, being formed at the catalytic site.
Borohydrides↗
Thiamine deficiency in patients with congestive heart failure receiving long-term furosemide therapy: a pilot study.
PURPOSE: To test the hypothesis that long-term furosemide therapy in patients with congestive heart failure (CHF) is associated with clinically significant thiamine deficiency via urinary loss. DESIGN: (1) Biochemical evaluation of thiamine status in hospitalized patients with CHF treated with long-term furosemide and in age-matched control patients. (2) Uncontrolled trial of the effect of intravenous thiamine on cardiac performance in a subset of six patients with CHF. SETTING: General medical ward of a teaching community hospital. PATIENTS: Twenty-three patients with chronic CHF receiving furosemide, and 16 age-matched control patients without heart failure and not taking diuretics. Daily furosemide doses were 80 to 240 mg, and duration of furosemide therapy was 3 to 14 months. Patients with identifiable causes of inadequate thiamine intake, absorption, or utilization or increased metabolic requirements were excluded. INTERVENTION: A 7-day course of intravenous thiamine, 100 mg twice daily, in six consenting patients with CHF. RESULTS: A high thiamine pyrophosphate effect (TPPE), indicating thiamine deficiency, was found in 21 of 23 furosemide-treated patients and in two of 16 controls (p less than 0.001). The mean (+/- SE) TPPE (normal: 0% to 15%) in furosemide-treated and control patients was 27.7 +/- 2.5% and 7.1 +/- 1.6%, respectively (p less than 0.001). Despite the high TPPE, the mean (+/- SE) urinary thiamine excretion in the furosemide-treated patients (n = 18) was inappropriately high (defined as greater than 130 micrograms/g creatinine), 410 +/- 95 micrograms/g creatinine, even in comparison with that in the controls (n = 14): 236 +/- 69 micrograms/g creatinine. In six patients treated with intravenous thiamine, the elevated TPPE decreased to normal, from a mean (+/- SE) of 27.0 +/- 3.8% to 4.5 +/- 1.3% (p less than 0.001), indicating normal thiamine utilization capacity. Left ventricular ejection fraction increased in four of five of these patients studied by echocardiography. CONCLUSIONS: These preliminary findings suggest that long-term furosemide therapy may be associated with clinically significant thiamine deficiency due to urinary loss and contribute to impaired cardiac performance in patients with CHF. This deficit may be prevented or corrected by appropriate thiamine supplements.
Adult↗
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.
Adenosine Diphosphate↗
Biochemical studies of pyrithiamine-resistant mutants of Escherichia coli K12.
The biochemical properties of strains (PT-R101 and PT-R108) of Escherichia coli K12 resistant to growth inhibition by pyrithiamine, and antimetabolite of thiamine, have been studied. Intracellular thiamine pyrophosphate concentration in these resistant strains was slightly but definitely higher than that in the parent strain. Thiamine synthesis from the pyrimidine and thiazole moieties of thiamine by cell suspensions was greater in the resistant strains than the parent strain. The activities of enzymes involved in thiamine biosynthesis in these pyrithiamine-resistant strains were 2-3 times higher than the parent strain (3301), except for thiamine-phosphate kinase, which was indetectable in in vitro assay of the activity. However, other evidence indicates that this enzyme is not defective but is functioning in vivo and, furthermore, that the negligible activity of this enzyme did not affect the growth rate of the mutants. The activities of these enzymes were further enhanced when PT-R101 was grown on 5mM adenine and were reduced almost to zero when the strain was grown on 0.1 muM thiamine in the same way as the parent strain. However, when these resistant strains were grown on a low concentration of thiamine such as 0,05 muM, thiamine synthesis by cell suspensions also decreased, but only to a limited extent compared with the parent strain. These results suggest that PT-R101 and PT-R108 are altered in the mechanisms of regulation of thiamine biosynthesis. Their altered properties might be due to a reduced binding affinity of the repressor protein, which is involved on the regulation of thiamine synthesis, for the corepressor, thiamine pyrophosphate,
Adenine↗
Inhibition of cell proliferation and dihydrofolate reductase by liposomes containing methotrexate-dimyristoylphosphatidylethanolamine derivatives and by the glycerophosphorylethanolamine analogs.
Liposomes, which were prepared with the three methotrexate (MTX)-dimyristoylphosphatidylethanolamine (DMPE) derivatives described in the preceding paper, were tested for their ability to block proliferation of mouse 3T3 and L1210 cells. Tritiated deoxyuridine incorporation into DNA could be completely inhibited by liposomes sensitized with MTX-DMPE I (MTX-gamma-DMPE). Under similar conditions, liposomes containing MTX-DMPE II (MTX-alpha-DMPE) and MTX-DMPE III (MTX-alpha, gamma-diDMPE) produced partial and no inhibition, respectively. These effects on cell growth were paralleled by the capacity of liposomes, prepared with each of the DMPE derivatives, to inhibit dihydrofolate reductase isolated from L1210 cells. Analogous experiments with the three corresponding glycerophosphorylethanolamine (glyceroPE) analogs also indicated that MTX-glyceroPE I was the most effective inhibitor of both cell proliferation and enzymatic activity. However, MTX-DMPE I sensitized liposomes apparently enter target cells as a consequence of phagocytosis, and not via the ubiquitous methotrexate transport system that is employed by MTX-glyceroPE I. For example, novel use of thiamine pyrophosphate showed that this compound had no influence on inhibition of cell proliferation due to liposomes, whereas thiamine pyrophosphate could completely antagonize the inhibitory effects of methotrexate and MTX-glyceroPE I. The results are discussed with reference to possible therapeutic advantages of these liposomes.
Ammonium Chloride↗
A constitutive thiamine metabolism mutation, thi80, causing reduced thiamine pyrophosphokinase activity in Saccharomyces cerevisiae.
We identified a strain carrying a recessive constitutive mutation (thi80-1) with an altered thiamine transport system, thiamine-repressible acid phosphatase, and several enzymes of thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. The mutant shows markedly reduced activity of thiamine pyrophosphokinase (EC 2.7.6.2) and high resistance to oxythiamine, a thiamine antagonist whose potency depends on thiamine pyrophosphokinase activity. The intracellular thiamine pyrophosphate content of the mutant cells grown with exogenous thiamine (2 x 10(-7) M) was found to be about half that of the wild-type strain under the same conditions. These results suggest that the utilization and synthesis of thiamine in Saccharomyces cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level.
Acid Phosphatase↗
Molecular analysis of abnormal pyruvate dehydrogenase in a patient with thiamine-responsive congenital lactic acidemia.
A patient who responded to thiamine therapy with reduction of lactate in the blood and cerebrospinal fluid and clinical improvement was studied. Cultured lymphoblastoid cells of this patient were found to show reduced activities of pyruvate dehydrogenase complex (PDHC) and pyruvate dehydrogenase, decreased affinity of PDHC for thiamine pyrophosphate, and defective activation of PDHC by pyruvate dehydrogenase phosphatase. PDHC deficiency in fibroblasts and biopsied muscle of this patient was also due to the decreased affinity of PDHC for thiamine pyrophosphate. A mutation in the E1 alpha subunit containing the thiamine binding site and serine phosphorylation site regulating the activation/inactivation of PDHC was characterized by the polymerase chain reaction and DNA sequencing. A single A-->G transition was identified at position 131, resulting in the substitution of Arg-44 for His-44. This mutation must be a de novo mutation because it was not found in either parent's genomic DNA. In this study, we have obtained the first evidence at the molecular level for a mutation of thiamine-responsive PDHC deficiency.
Acidosis, Lactic↗
Thiamine, riboflavin, folate, and vitamin B12 status of low birth weight infants receiving parenteral and enteral nutrition.
Thirty infants were randomly assigned to receive either 3 mL of MVI-Pediatric supplement (PAR3 group, parenterally fed) or 2 mL (PAR2 group, parenterally fed). For the first week, 100% received total parenteral nutrition (TPN), 50% by the second, and less than 33% by the third. Eighteen control infants received enteral feeds of infant formula. Baseline (before TPN) and subsequent weekly blood samples, dietary data, and 24-hour urine collections were obtained. The adequacies of thiamine and riboflavin were assessed by the thiamine pyrophosphate effect and erythrocyte glutathione reductase activity, respectively. Urinary thiamine and riboflavin levels were measured by fluorometry. Plasma folate, red blood cell folate, urinary folate, and plasma vitamin B12 concentrations were determined by radioassay. No differences between groups were observed in thiamine pyrophosphate effect, erythrocyte glutathione reductase activity, urinary B1 or B2, or red blood cell folate levels at any time. Plasma folate differed (p less than .05) among the PAR3 group (24 +/- 7 ng/mL), and both the PAR2 (13 +/- 5 ng/mL) and enterally fed (ENT) groups (16 +/- 3 ng/mL) before the initiation of feeds, at week 1 (PAR3 = 32 +/- 15 ng/mL; PAR2 = 18 +/- 4 ng/mL; ENT = 19 +/- 9, ng/mL) and between the PAR3 (30 +/- 16 ng/mL) and PAR2 (16 +/- 4 ng/mL) infants at week 2. Plasma vitamin B12 levels differed among the ENT groups (551 +/- 287 pg/mL) and both the parenteral groups (PAR2 = 841 +/- 405 pg/mL; PAR3 = 924 +/- 424 pg/mL) at week 1 and between the ENT (530 +/- 238 pg/mL) and PAR3 (999 +/- 425 pg/mL) groups at week 2.(ABSTRACT TRUNCATED AT 250 WORDS)
Birth Weight↗
Blood thiamine and thiamine phosphate ester concentrations in alcoholic and non-alcoholic liver diseases.
Thiamine state was investigated in patients with alcoholic liver disease, patients with various non-alcoholic liver diseases, and controls using a direct technique (thiochrome assay) to measure thiamine, thiamine monophospate, and the active coenzyme thiamine pyrophosphate in whole blood after isolating the fractions by ion exchange chromatography. Overall nutrition was similar in all groups as assessed by anthropometry, and no patient had clinical evidence of thiamine deficiency. There was no significant difference among the groups in mean concentration of any form of thiamine. The scatter was much greater in patients with alcoholic liver disease but only 8.7% had biochemical thiamine deficiency (defined as a blood concentration of the active coenzyme greater than 2 SD below the mean control value). An unexpected finding was of abnormally high total thiamine concentrations (greater than 2 SD above the mean control value) in 17.4% of patients with alcoholic liver disease, the highest concentrations being found in two patients with severe alcoholic hepatitis and cirrhosis. The ratio of phosphorylated to unphosphorylated thiamine was calculated as an index of phosphorylation and, although the mean did not differ significantly among the groups, the range was greatest in alcoholic liver disease. The lowest ratios occurred in the two patients with severe alcoholic hepatitis, but neither had evidence of thiamine pyrophosphate deficiency. Contrary to studies using indirect assay techniques, these results suggest that thiamine deficiency is unusual in well nourished patients with alcoholic liver disease. The new finding of unexpectedly high thiamine concentrations in some patients may be due to abnormalities of hepatic storage or release in liver disease, particularly in severe alcoholic hepatitis. There was no convincing evidence of impaired thiamine phosphorylation in any patients with liver disease. Conclusions from studies using indirect assays on the prevalence and mechanisms of thiamine deficiency in liver diseases may not be valid.
Adult↗