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PubMed1995

Molecular genetics of transketolase in the pathogenesis of the Wernicke-Korsakoff syndrome.

Thiamine deficiency, a frequent complication of alcoholism, plays an important role in the pathogenesis of the Wernicke-Korsakoff syndrome [WKS]. Previous work by a number of investigators has implicated the thiamine-utilizing enzyme transketolase [Tk] as being involved mechanistically in the genetic predisposition to WKS. In particular, Tk derived from fibroblasts has been found to have an increased Km app for its cofactor thiamine pyrophosphate [TPP] and/or exist in different isoelectric forms in alcoholic patients with WKS as compared with unaffected individuals. We have demonstrated that these differences are not due to different Tk alleles, tissue-specific Tk isozymes, or differential mRNA splicing. These findings point to other mechanisms to explain the biochemical Tk variants, such as differences in assembly of the functional holoenzyme or differences in modification of the primary translation product. Tk assembly or modification, once biochemically characterized, may be found to be subject to genetic variation.

Binding Sites↗

PubMed1988

Elucidation of the 2-aminoethylphosphonate biosynthetic pathway in Tetrahymena pyriformis.

The biosynthetic reaction pathway leading to the natural product, 2-aminoethylphosphonate in Tetrahymena pyriformis has been elucidated. Incubation of [32P]PEP and [14C]PEP with T.pyriformis cellular homogenate fortified with Mg2+ and alanine/pyridoxal phosphate, yielded 2-aminoethylphosphonate as the minor reaction product (2-5% yield) and phosphoglycerate and pyruvate plus orthophosphate as the major products. Inclusion of thiamine pyrophosphate in the reaction mixture increased the yield of 2-aminoethylphosphonate by a factor of 10. Incubation of phosphonoacetaldehyde or phosphonopyruvate in the cellular homogenate also provided 2-aminoethylphosphonate. The cellular homogenate catalyzed the transformation of phosphonoacetaldehyde to 2-aminoethylphosphonate in an ca. 80% yield. However, the maximum yield of 2-aminoethylphosphonic acid obtained by use of phosphonopyruvate was only 15%. The major reaction pathways induced by treatment of phosphonopyruvate with the cellular extract involved its competitive conversion to PEP and pyruvate plus orthophosphate.

Acetaldehyde↗

PubMed1986

Transketolase abnormality in tolazamide-induced Wernicke's encephalopathy.

We studied a thiamine-dependent enzyme, transketolase, from fibroblasts of a diabetic patient who developed Wernicke's encephalopathy when treated with tolazamide, in order to delineate if this patient also had transketolase abnormality [high Km for thiamine pyrophosphate (TPP)], as previously reported in postalcoholic Wernicke-Korsakoff syndrome. In addition to this patient, we also studied this enzyme from three diabetic kindreds without any history of Wernicke's encephalopathy and from four normal controls. We found that the above-mentioned patient and one of the diabetic kindreds with no history of Wernicke's encephalopathy had abnormal transketolase as determined by its Km for TPP. These data suggest a similarity between postalcoholic Wernicke-Korsakoff syndrome and the patient with tolazamide-induced Wernicke's encephalopathy from the standpoint of transketolase abnormality.

Fibroblasts↗

PubMed1995

The effect of phosphorylation on pyruvate dehydrogenase.

Phosphorylation of the pyruvate dehydrogenase component (E1) of the muscle pyruvate dehydrogenase complex (PDC) by E1-kinase inhibits substrate conversion both in oxidative and non-oxidative reactions. Circular dichroism spectra were used to monitor the effect of phosphorylation on the following stages of the process: holoform formation from apo-E1 and thiamine pyrophosphate (TPP), substrate binding and active site deacetylation. It has been shown that phosphorylation of E1 reduces its affinity for TPP and prevents holo-E1 interaction with pyruvate. Phosphorylated and dephosphorylated PDC convert 2-hydroxyethyl-TPP in similar ways involving half of their active sites; all active sites of E1 function in the presence of deacetylating agents. The data obtained suggest that the phosphorylation and substrate binding sites interact with each other.

Adenosine Triphosphate↗

PubMed1980

[Formation of oligomeric form of transketolase and the factors influencing this process].

The immobilized transketolase (dimer) is a mixture of two enzyme forms, i. e. a completely active and completely inactive ones. The decomposition of the active enzyme form into subunits to form immobilized monomers occurs at a higher rate than that of the inactive form. The reassociation of the monomers with a formation of dimers strongly depends on pH, the pH optimum of reassociation being equal to 7,9--9,2. Thiamine pyrophosphate and Ca2+ as well as Ca2+ alone but taken at sufficiently high concentrations favours the association of the monomers into a dimer. The reassociated dimers exhibit a complete catalytic activity only when the reassociation occurs in the presence of Ca2+. The activity of reassociated dimers obtained in the absence of Ca2+ was decreased by 50%. The reassociation capacity of the immobilized subunits obtained in the absence of beta-mercaptoethanol was considerably decreased. The immobilized subunits obtained in the presence of beta-mercaptoethanol and then treated by N-ethylmaleimide were not capable of reassociation.

Calcium↗

PubMed1988

Western blotting assay of transketolase concentration in human hemolysates.

Using a rabbit anti-human transketolase antiserum and Western blotting we can determine nanogram amounts of transketolase in human hemolysates quantitatively. Transketolase concentration in 18 apparently healthy subjects was 55.7 +/- 12.1 micrograms/g Hb (mean +/- SD). Transketolase concentration correlated positively with the enzyme activity both with and without in vitro addition of thiamin pyrophosphate. However, the former had a closer correlation (r = 0.8418, P less than 0.001) than the latter (r = 0.6703, P less than 0.01). A heavy drinker with an extremely low transketolase activity had proportionally low concentration to the activity. These results indicate that transketolase in hemolysates, whether it is holoenzyme or apoenzyme activated in vitro, has an identical specific activity among all subjects studied and that the reduced activity of transketolase in alcoholics is due to the reduced content of the enzyme protein. This method is applicable to study the dynamics and the abnormality of apotransketolase in human hemolysates.

Alcohol Drinking↗

PubMed2001

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases.

This review summarizes the recent developments on the regulation of human pyruvate dehydrogenase complex (PDC) by site-specific phosphorylation by four kinases. Mutagenic analysis of the three phosphorylation sites of human pyruvate dehydrogenase (E1) showed the site-independent mechanism of phosphorylation as well as site-independent dephosphorylation of the three phosphorylation sites and the importance of each phosphorylation site for the inactivation of E1. Both the negative charge and size of the group introduced at site 1 were involved in human E1 inactivation. Mechanism of inactivation of E1 was suggested to be site-specific. Phosphorylation of site 1 affected E1 interaction with the lipoyl domain of dihydrolipoamide acetyltransferase, whereas phosphorylation site 3 appeared to be closer to the thiamine pyrophosphate (TPP)-binding region affecting coenzyme interaction with human E1. Four isoenzymes of pyruvate dehydrogenase kinase (PDK) showed different specificity for the three phosphorylation sites of E1. All four PDKs phosphorylated sites 1 and 2 in PDC with different rates, and only PDK1 phosphorylated site 3. PDK2 was maximally stimulated by the reduction/acetylation of the lipoyl groups of E2. Presence of the multiple phosphorylation sites and isoenzymes of PDK is important for the tissue-specific regulation of PDC under different physiological conditions.

Acetylation↗

PubMed1973

The metabolism of nitrilotriacetate by a pseudomonad.

1. An organism that grows on nitrilotriacetate as sole source of carbon and energy was isolated in pure culture and was identified as a pseudomonad. 2. Cell-free extracts of the nitrilotriacetate-grown pseudomonad contain an enzyme that catalyses the NADH-and O(2)-dependent oxidation of nitrilotriacetate to iminodiacetate and glyoxalate. This enzyme is absent from extracts of glucose-grown cells. 3. Compared with growth on glucose, growth on nitrilotriacetate results in increased activities of enzymes of glycine and serine metabolism, namely serine hydroxymethyltransferase, glycine decarboxylase, serine-oxaloacetate aminotransferase and hydroxypyruvate reductase. 4. Cell-free extracts of the nitrilotriacetate-grown organism contain the enzyme glyoxalate carboligase and, when supplemented with NADH, Mg(2+) and thiamin pyrophosphate, can catalyse the anaerobic conversion of glyoxalate into glycerate. 5. These results are incorporated in a scheme which shows the oxidative metabolism of nitrilotriacetate by the successive removal of C(2) units to form 2mol of glyoxalate and 1mol of glycine per mol of nitrilotriacetate degraded. The glyoxalate and glycine are then both metabolized to glycerate by separate pathways, via tartronic semialdehyde and serine respectively. The role of this scheme in the growth of the organism on nitrilotriacetate is discussed.

Acetates↗

PubMed1986

Inhibition of pyruvate dehydrogenase complex by moniliformin.

The mechanism for the inhibition of pyruvate dehydrogenase complex from bovine heart by moniliformin was investigated. Thiamin pyrophosphate proved to be necessary for the inhibitory action of moniliformin. The inhibition reaction was shown to be time-dependent and to follow first-order and saturation kinetics. Pyruvate protected the pyruvate dehydrogenase complex against moniliformin inactivation. Extensive dialysis of the moniliformin-inactivated complex only partially reversed inactivation. Moniliformin seems to act by inhibition of the pyruvate dehydrogenase component of the enzyme complex and not by acting on the dihydrolipoamide transacetylase or dehydrogenase components, as shown by monitoring the effect of moniliformin on each component individually. On the basis of these results, a suicide inactivator mechanism for moniliformin on pyruvate dehydrogenase is proposed.

Cyclobutanes↗

PubMed2002

Characterization of a missense mutation at histidine-44 in a pyruvate dehydrogenase-deficient patient.

Genetic defects in pyruvate dehydrogenase complex (PDC) cause lactic acidosis, neurological deficits, and often early death. Most mutations of PDC are localized in the alpha subunit of the pyruvate dehydrogenase (E1) component. We have kinetically characterized a patient's missense mutation alphaH44R in E1alpha by creating and purifying three recombinant human E1s (alphaH44R, alphaH44Q, and alphaH44A). Substitutions at histidine-15 resulted in decreased V(max) values (6% alphaH44R; 30% alphaH44Q; 90% alphaH44A) while increasing K(m) values for thiamine pyrophosphate (TPP) compared to wild-type (alphaH44R, 3-fold; alphaH44Q, 7-fold; alphaH44A, 10-fold). This suggests that the volume of the residue at site 15 is important for TPP binding and substitution by a residue with a longer side chain disrupts the active site more than the TPP binding site. The rates of phosphorylation and dephosphorylation of alphaH44R E1 by E1-kinase and phospho-E1 phosphatase, respectively, were similar to that of the wild-type E1 protein. These results provide a biochemical basis for altered E1 function in the alphaH44R E1 patient.

Acetyltransferases↗

PubMedDate not supplied

[Effect of preliminarily administered aspartate to the body and its combination with a vitamin-coenzyme complex on the catabolism of L-[14C]-aspartate in tissues of various mouse organs in hermetically closed space].

Effect of factors of sealed non-aired space on the organism leads to the enhancement of catabolism of L-4-[14C]-aspartate in the mice encephalon to 14CO2. Preliminary administration of L-aspartate to the organism (100 mg/kg) leads to the intensification of these adaptative reactions and prolongs life of animals under these conditions. Accumulation of the introduced aspartate in the liver and encephalon gets more intensive and its supply to the blood is more rapid under these conditions. Simultaneous (together with L-aspartate) administration of the vitamin-coenzyme complex (pentapyruvate) which includes thiamine pyrophosphate, lipoate, sodium 4-phosphopantotenate, nicotinate and riboflavin-mononucleotide and stimulates the function of the key links of the Krebs cycle to animals has induced intensification of the protective effect of L-aspartate and evoked further activation of the L-aspartate catabolism in the encephalon, mainly at late, preagonal stages of the developing pathological state. The data presented confirm that protective effect of L-aspartate, provided the effect of the closed space factors on the organism, is a result of its quick introduction, into energy mebolism of tissues, in particular, of the nervous tissue.

Animals↗

PubMed1985

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex.

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.

Acetylation↗

PubMed2005

Roles of Mg2+ in TPP-dependent riboswitch.

We quantified the effect of Mg(2+) on thiamine pyrophosphate (TPP) binding to TPP-dependent thiA riboswitch RNA. The association constant of TPP binding to the riboswitch at 20 degrees C increased from 1.2 x 10(6) to 50 x 10(6) M(-1) as the Mg(2+) concentration increased from 0 to 1 mM. Furthermore, circular dichroic spectra under various conditions showed that 1 mM Mg(2+) induced a local structural change of the riboswitch, which might be pivotal for TPP binding. These results indicate that a physiological concentration of Mg(2+) can regulate TPP binding to the thiA riboswitch.

5' Untranslated Regions↗

PubMed1986

Targeting, internalization, and cytotoxicity of methotrexate-monoclonal anti-stage-specific embryonic antigen-1 antibody conjugates in cultured F-9 teratocarcinoma cells.

Methotrexate (MTX) conjugates of a monoclonal antibody, anti-SSEA-1, containing an average of 45 mol MTX/mol of immunoglobulin M, were prepared by a carbodiimide coupling reaction. Binding experiments indicate that conjugation does not decrease the affinity of the antibody for its antigen. The conjugate strongly inhibits the growth of SSEA-1-bearing F-9 teratocarcinoma cells, with 50% inhibitory dose of 4.5 nM MTX, which makes it more active than free MTX (50% inhibitory dose of 15 nM). The drug-free antibody is not cytotoxic to F-9 cells at the concentrations used. The high efficacy of the conjugated drug may be due in part to the fact that anti-SSEA-1 antibody is an immunoglobulin M. MTX conjugated to nonspecific immunoglobulin M has little inhibitory effect (50% inhibitory dose of 150 nM). When acting on SSEA-1 negative cells, the two conjugates have only a small but identical effect. Thiamine pyrophosphate, an inhibitor of MTX transport, can prevent the cytotoxicity of the free MTX but not that of the anti-SSEA-1 conjugate. Leupeptin, an inhibitor of lysosomal protease, can partially protect F-9 cells against the antibody conjugate but not against free MTX. These results indicate that the MTX antibody conjugate binds specifically to F-9 cells, and is internalized and intracellularly degraded to release a small molecular active drug. Pretreatments of F-9 cells for 1 h with unlabeled antibody inhibits the subsequent uptake of identical concentration of labeled conjugate. The rate of internalization, however, regains almost normal values within 4 h, indicating a rapid reappearance of free antigenic sites at the cell surface.

Animals↗

PubMed1980

An evaluation of a modified erythrocyte transketolase assay for assessing thiamine nutritional adequacy.

Transketolase (TK) activity was assessed by incubating hemolyzed whole blood with ribose-5-phosphate both in the presence (TKsat.) and in the absence (TK) of thiamine pyrophosphate (TPP). The amount of pentoses utilized between 5 and 60 min after the start of the incubation was taken as a measure of the TK activity. The TPP effect was expressed at (TKsat. - TK)/TK x 100. The coefficient of variation for the TPP effect determination was 9.6% within the TPP effect range from 6 to 22%. Within the TPP effect range from 24 to 46%, the coefficient of variation was 8.7%. The mean TPP effect as measured in a group of healthy individuals amounted to 13.5%, with a range from 8.3 to 18.5%. In a group of obese women submitted to an energy-restricted diet, the TPP effect ranged from 12.7 to 30%. In patients suffering from Wernicke's encephalopathy, the TPP effect varied from 28 to 67%.

Clinical Enzyme Tests↗

PubMedDate not supplied

[Isolation and characterization of a membrane-bound pyruvate dehydrogenase complex from the phototrophic bacterium Rhodospirillum rubrum (author's transl)].

The pyruvate dehydrogenase complex from the photosynthetic bacterium Rhodospirillum rubrum was associated with the membrane fraction both in heterotrophically and photosynthetically grown cells. The complex was separated from the membranes and partially purified by precipitation with MgSO4 and gelfiltration through Sepharose 4B. The purified complex had a specific activity of 1.5-2mumol/min-mg protein and contained the following partial activities: pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoamide transacetylase (EC 2.3.1.12) and dihydrolipoamide dehydrogenase (EC 1.6.4.3). Contrary to other bacterial pyruvate dehydrogenase complexes, the enzyme complex from R. rubrum revealed no cooperatively between pyruvate binding sites. The kinetic constants (Km) for the overall reaction were (in mM): 0.14 (pyruvate), 0.07 (NAD) and 0.025 (coenzyme A). The Km for thiamine pyrophosphate was dependent on the nature and the concentration of the divalent metal ion (Mn or Mg) present in the reaction mixture, the values ranging from 0.5 to 3 micrometer. NADH was a potent inhibitor (Ki=5 micrometer) of the enzyme complex and the dihydrolipo amide dehydrogenase. The inhibition was competitive with respect to NAD. In addition to its rapid inhibitory effect, NADH also inactivated the enzyme. Cysteine partially protected the enzyme complex against NADH-inactivation. Acetyl-coenzyme A also inhibited the overall reaction (Ki=40 micrometer). The inhibition was dependent on the concentration of coenzyme A, but independent of the concentration of pyruvate. Sugar phosphates, phosphoenolpyruvate, citric acid cycle intermediates and nucleosidephosphates (1 mM) had no pronounced effect on the overall reaction.

Acetyl Coenzyme A↗

PubMed2006

Structure of the thiazole biosynthetic enzyme THI1 from Arabidopsis thaliana.

Thiamin pyrophosphate is an essential coenzyme in all organisms that depend on fermentation, respiration or photosynthesis to produce ATP. It is synthesized through two independent biosynthetic routes: one for the synthesis of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (pyrimidine moiety) and another for the synthesis of 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate (thiazole moiety). Herein, we will describe the three-dimensional structure of THI1 protein from Arabidopsis thaliana determined by single wavelength anomalous diffraction to 1.6A resolution. The protein was produced using heterologous expression in bacteria, unexpectedly bound to 2-carboxylate-4-methyl-5-beta-(ethyl adenosine 5-diphosphate) thiazole, a potential intermediate of the thiazole biosynthesis in Eukaryotes. THI1 has a topology similar to dinucleotide binding domains and although details concerning its function are unknown, this work provides new clues about the thiazole biosynthesis in Eukaryotes.

Adenosine Triphosphate↗

PubMed1988

Red cell transketolase studies in a private practice specializing in nutritional correction.

Erythrocyte transketolase (TKA) and thiamin pyrophosphate percentage uptake, or effect (TPPE) were performed on 1011 patients between 1983 and 1986. The subjects were drawn from a private practice specializing in nutritional correction as a major therapeutic thrust. Either TKA or TPPE or both were abnormal in 283 (28%). Out of the total number of patients with these abnormal studies 36 have been selected as representative. Their clinical presentation is correlated with subsequent laboratory testing in order to draw attention to the practical value of the test in a clinical setting. These data are presented because of the surprisingly high incidence of abnormal tests, that strongly suggests that there is widespread marginal nutritional deficiency in the United States and that it represents a clinical problem that deserves far more attention than it is presently receiving.

Adolescent↗