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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↗

PubMed1987

A comparison of transketolase assay and transketolase and lactate dehydrogenase activity levels in whole blood and red cell hemolysates and in leukocytes.

1. A study was made of transketolase activity in red and white blood cells and of conditions for assay for transketolase activity and for assessment of the "TPP effect" in human and rat blood. 2. The ratio of the transketolase activity in white cells to that in red cells varied between 23 and 93. 3. Red cells or white cells can both be used for assessment of transketolase activity and the "TPP effect", but the best source for evaluation of transketolase activity and the percent change on addition of thiamin diphosphate appears to be whole blood.

Animals↗

PubMed1997

Analysis of a transketolase gene from Kluyveromyces lactis reveals that the yeast enzymes are more related to transketolases of prokaryotic origins than to those of higher eukaryotes.

The role of the pentose-phosphate pathway in carbohydrate metabolism of the yeast Kluyveromyces lactis, and the evolutionary relationships between the encoding genes, was investigated. For this purpose, we isolated the gene encoding transketolase (KlTKL1) and determined its nucleotide sequence. Surprisingly, comparisons of the deduced amino-acid sequence with those from other organisms revealed that the yeast enzymes are more related to those from prokaryotic sources than to those from higher eukaryotes. Functional analyses showed that KlTKL1 also complemented a Saccharomyces cerevisiae tkl1 tkl2 double mutant for growth in the absence of aromatic amino acids and restored transketolase activity in this mutant. A band detected in these transformants by Western-blot analysis corresponded to a band detected in K. lactis both in a wild-type strain and in a multicopy transformant with elevated transketolase activity.

Amino Acid Sequence↗

PubMed1988

Isolation of transketolase from rabbit liver and comparison of some of its kinetic properties with transketolase from other sources.

1. Rabbit liver transketolase activity was purified 56-fold using the following steps: ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-25, concentration through an Amicon ultrafiltration cell and rechromatography on DEAE-Sephadex A-25. 2. The enzyme showed an optimum PH for activity at 7.8-8.0. 3. The optimum temperature was around 40 degrees C and the activation energy calculated from the Arrhenius plot was found to be 11.4 kcal/mole. 4. The molecular weight of the enzyme, as determined by gel filtration, was found to be approximately 162,000, while the content of thiamin diphosphate was between 1.8 and 2 mumole per mole protein. 5. Addition of thiamin diphosphate and magnesium chloride did not influence the activity. 6. From the kinetic studies of the enzyme, the Km values for xylulose-5-phosphate, ribose-5-phosphate and fructose-6-phosphate were 3.8 x 10(-5) M, 9.5 x 10(-5) M and 1.1 x 10(-2) M, respectively.

Animals↗

PubMed1985

Assessment of the thiamine nutritional status. An evaluation of erythrocyte transketolase activity, the stimulated erythrocyte transketolase activity, and the thiamine pyrophosphate effect.

The most widely accepted approach to estimation of thiamine nutrition has been the measurement of the erythrocyte transketolase activity (ETKA), the ETKA stimulated in vitro with thiamine pyrophosphate (TPP) (which is suggested to be designated ETKAS) and the percentage increase of ETKA after stimulation with TPP in vitro, called the thiamine pyrophosphate effect (TPPE). In spite of 30 years of experience in the determination of these variables doubtfulness still exists of how to assess them. On the basis of a study of a group of alcoholics and a group of non-alcoholics a system of evaluation of ETKA, ETKAS and TPPE is proposed.

Adult↗

PubMed2005

Mutations in the transketolase-like gene TKTL1: clinical implications for neurodegenerative diseases, diabetes and cancer.

Transketolase proteins or transketolase enzyme activities have been related to neurodegenerative diseases, diabetes, and cancer. Transketolase enzyme variants and reduced transketolase enzyme activities are present in patients with the neurodegenerative disease Wernicke-Korsakoff syndrome. In Alzheimer's disease patients transketolase protein variants with different isoelectric points or a proteolytic cleavage leading to small transketolase protein isoforms have been identified. In diabetes mellitus patients reduced transketolase enzyme activities have been detected and the lipid-soluble thiamine derivative benfotiamine activates transketolase enzyme reactions, thereby blocking three major pathways of hyperglycemic damage and preventing diabetic retinopathy. In cancer inhibition of transketolase enzyme reactions suppresses tumor growth and metastasis. All the observed phenomena have been interpreted solely on the basis of a single transketolase gene (TKT) encoding a single transketolase enzyme. No mutations have been identified so far in TKT transketolase explaining the altered transketolase proteins or transketolase enzyme activities found in neurodegenerative diseases, diabetes and cancer. We demonstrate the presence of a second transketolase enzyme (TKTL1) in humans. During the evolution of the vertebrate genome, mutations in this transketolase gene (TKTL1) have led to tissue-specific transcripts different in size, which encode an enzymatically active transketolase protein as well as different smaller protein isoforms. The mutations within the TKTL1 gene caused a mutant transketolase enzyme with an altered substrate specificity and reaction modus. Here we characterize the TKTL1 gene and its encoded TKTL1 protein(s) and discuss the medical and clinical implications of this mutated transketolase. We furthermore postulate a novel metabolic concept for the understanding, prevention and therapy of neurodegenerative diseases, diabetes and cancer.

Diabetes Mellitus↗

PubMed1998

Properties and functions of the thiamin diphosphate dependent enzyme transketolase.

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer's disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.

Amino Acid Sequence↗

PubMed1997

Molecular evolutionary analysis of the thiamine-diphosphate-dependent enzyme, transketolase.

Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade.

Amino Acid Sequence↗

PubMed1996

Amplification of the transketolase gene in desensitization-resistant mutant Y1 mouse adrenocortical tumor cells.

As shown previously, mutants of the Y1 mouse adrenocortical tumor cell line that resist agonist-induced desensitization of adenylyl cyclase have elevated levels of a 68-kDa protein (designated p68), suggesting a possible relationship between p68 and the regulation of adenylyl cyclase activity. In the present study, cDNA cloning and sequencing were used to identify p68 as mouse transketolase. Cells overexpressing p68 exhibited a 17.4-fold increase in transketolase enzymatic activity relative to parental Y1 cells and a 28-fold amplification of the transketolase gene as determined by Southern blot hybridization analysis. Using fluorescent in situ hybridization analysis, the transketolase gene was mapped to mouse chromosome 16B1 and to human chromosome 3p21.2. Transketolase gene amplification was associated with telomeric fusion of the chromosome 16 pair together with the appearance of multiple copies of the transketolase gene throughout a different chromosome. The relationship between overexpression of transketolase and desensitization resistance was evaluated in somatic cell hybrids formed between a desensitization-resistant adrenal cell line and a desensitization-sensitive rat glial cell line. In these hybrids, transketolase overexpression behaved dominantly, whereas desensitization resistance behaved recessively. These results dissociate the desensitization resistance phenotype from overexpression of transketolase and suggest that desensitization resistance may have resulted from disruption of an essential regulatory gene in conjunction with the amplification event.

Adenylyl Cyclases↗

PubMed1995

[Some properties of multiple forms of transketolase from baker's yeast].

It has been shown that transketolase A does not differ from the enzyme earlier described in the literature by a number of properties (lack of intersubunit disulfide bonds, identical number of sulfhydryl groups, two values of Km for thiamine pyrophosphate and identity of their absolute values). Transketolase C subunits are linked together by disulfide bonds; their total number in the enzyme molecule is five (transketolase C-1) or six (transketolase C-2). The Km values of transketolase C-1 for thiamine pyrophosphate are commensurate with those of transketolase A. Transketolase C-2 has only one Km value for thiamine pyrophosphate which is close to one of the two Km values for transketolase A. The maximal rate values for transketolases C-1 and C-2 with dihydroxyacetone as substrate differ by more than one order of magnitude.

Disulfides↗

PubMed2002

Effects of furazolidone, PCB77, PCB126, Aroclor 1248, paraquat and p,p'-DDE on transketolase activity in embryonal chicken brain.

The effect of in ovo exposure to PCBs, DDE and paraquat on transketolase activity was measured in 19-day-old chicken embryos. Furazolidone was used as a positive control for decreased activity of the enzyme. The potency of contaminants to interact with transketolase was also tested in an in vitro system, using control brain 7000xg supernatants containing the enzyme. No effects were found on transketolase activity after in ovo or in vitro exposure to PCB126, Aroclor, DDE or paraquat. PCB77 decreased transketolase activity in vitro, but only at concentrations that, extrapolated to in ovo exposure, would be lethal to the embryo. Furazolidone decreased transketolase activity both in ovo and in vitro. For this contaminant, thiamine residues were analysed in the yolk sacs, but no differences were found between exposed and non-exposed eggs. Transketolase is dependent on thiamine pyrophosphate as a cofactor, and therefore, the decreased enzyme activity could be the result of an interaction between furazolidone and thiamine metabolism. Since thiamine residues were not affected by furazolidone and transketolase inhibition in vitro was similar to the inhibition after in ovo exposure, it was concluded that furazolidone interacted with transketolase on the enzymatic level rather than by a depletion of thiamine.

Animals↗

PubMed2005

In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.

Animals↗

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↗

PubMed1980

Glycolate formation catalyzed by spinach leaf transketolase utilizing the superoxide radical.

A homogeneous preparation of transketolase was obtained from spinach leaf; the specific enzyme activity was 9.5 mumolo of glyceraldehyde-3-P formed (mg of protein)-1 min-1, when xylulose-5-P and ribose-5-P were used as the donor and acceptor, respectively, of the ketol residue. Transketolase catalyzed the formation of glycolate from fructose-6-P coupled with the O2- -generating system of xanthine-xanthine oxidase. The addition of superoxide dismutase (145 units) or 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (5 mM), both O2- scavengers, to the reaction system inhibited glycolate formation 72 and 58%, respectively. The reacton was not inhibited by catalase. Mannitol, an .OH scavenger, and beta-carotene and 1,4-diazobicyclo[2.2.2]octane, 1O2 scavengers, showed little or no inhibitory effects. The rate of glycolate formation catalyzed by the transketolase system was measured in a coupled reaction with a continuous supply of KO2 dissolved in dimethyl sulfoxide, used as an O2- -generating system. The optimum pH of the reaction was above pH 8.5. The second-order rate constant for the reaction between transketolase and O2-, determined by the competition for O2- between nitroblue tetrazolium (NBT) and transketolase, was 1.0 X 10(6) M-1 s-1. Transketolase showed an inhibitory effect on the O2- -dependent reduction of NBT only if the reaction mixture was previously incubated with ketol donors such as fructose-6-P, xylulose-5-P, or glycolaldehyde. The results suggest the possibility that transketolase catalyzes O2- -dependent glycolate formation under increased steady-state levels of O2- in the chloroplast stroma.

Glycolates↗

PubMed2006

Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted.

Tumours ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumours and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumours leads to enhanced, oxygen-independent glucose usage and a lactate-based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumour growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualised cancer therapy based on the determination of metabolic changes in tumours that might enable the targeted inhibition of invasion and metastasis.

Adenocarcinoma↗

PubMed1989

Transketolase from human leukocytes. Isolation, properties and induction of polyclonal antibodies.

Transketolase has been purified for the first time from human leukocytes, according to a new procedure which consists of three conventional steps. The enzyme was finally detached from CM-cellulose by specific elution with a D-xylulose-5-phosphate/D-ribose-5-phosphate mixture and the isolated product exhibited a specific activity of about 10 units/mg protein at 37 degrees C. Transketolase preparations are contamination-free, except for a slight residual activity of phosphohexose isomerase. Kinetic constants for D-xylulose 5-phosphate and D-ribose 5-phosphate were found to be 0.19 mM and 0.63 mM, respectively. Pure transketolase migrates on SDS/PAGE as a single band, with a molecular mass of about 66 kDa. The isoelectrophoretic heterogeneity of transketolase was assessed either by activity staining or immunovisualization with anti-transketolase antisera, previously induced in rabbits. These techniques yielded two practically overlapping patterns consisting of 6-8 distinct bands within a pI range of 6.5-8.5. Both pure and crude transketolase preparations showed a similar heterogeneous profile, thus confirming the stability of the enzyme throughout purification. The occurrence of multiple enzyme forms in fresh human white cells has also been established by the analysis of transketolase in isolated populations of either lymphocytes or polymorphonuclear leukocytes, from individual healthy subjects.

Antigen-Antibody Reactions↗

PubMed1993

TKL2, a second transketolase gene of Saccharomyces cerevisiae. Cloning, sequence and deletion analysis of the gene.

Transketolase activity is indispensable for the generation of erythrose 4-phosphate and therefore necessary for the biosynthesis of the aromatic amino acids. Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells. Here we describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL2 demonstrates 71% identity with TKL1 [Sundström, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity. Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti-transketolase serum in Western blot analysis. In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids.

Amino Acid Sequence↗

PubMed1993

Cloning of human transketolase cDNAs and comparison of the nucleotide sequence of the coding region in Wernicke-Korsakoff and non-Wernicke-Korsakoff individuals.

Variants of the enzyme transketolase which possess reduced affinity for its cofactor thiamine pyrophosphate (high apparent Km) have been described in chronic alcoholic patients with Wernicke-Korsakoff syndrome. Since the syndrome has been shown to be directly related to thiamine deficiency, it has been hypothesized that such transketolase variants may represent a genetic predisposition to the development of this syndrome. To test this hypothesis, human transketolase cDNA clones were isolated, and their nucleotide and predicted amino acid sequence were determined. Transketolase was found to be a single copy gene which produces a single mRNA of approximately 2100 nucleotides. Additionally, the nucleotide sequence of the transketolase coding region in fibroblasts derived from two Wernicke-Korsakoff (WK) patients was compared to that of two nonalcoholic controls. Although nucleotide and predicted amino acid differences were detected between fibroblast cultures and the original cDNAs and among the cultures themselves, no specific nucleotide variations, which would encode a variant amino acid sequence, were associated exclusively with the coding region from WK patients. Thus, allelic variants of the transketolase gene cannot account for the biochemically distinct forms of the enzyme found in these patients nor be considered as a mechanism for genetic predisposition to the development of Wernicke-Korsakoff syndrome. Instead, the underlying mechanism must be extragenic and may be a result of differences in post-translational processing/modification of the transketolase polypeptide.

Alcohol Amnestic Disorder↗