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1.
Int J Mol Sci ; 24(3)2023 Jan 20.
Article in English | MEDLINE | ID: mdl-36768400

ABSTRACT

Transketolase catalyzes the interconversion of keto and aldo sugars. Its coenzyme is thiamine diphosphate. The binding of keto sugar with thiamine diphosphate is possible only after C2 deprotonation of its thiazole ring. It is believed that deprotonation occurs due to the direct transfer of a proton to the amino group of its aminopyrimidine ring. Using mass spectrometry, it is shown that a water molecule is directly involved in the deprotonation process. After the binding of thiamine diphosphate with transketolase and its subsequent cleavage, a thiamine diphosphate molecule is formed with a mass increased by one oxygen molecule. After fragmentation, a thiamine diphosphate molecule is formed with a mass reduced by one and two hydrogen atoms, that is, HO and H2O are split off. Based on these data, it is assumed that after the formation of holotransketolase, water is covalently bound to thiamine diphosphate, and carbanion is formed as a result of its elimination. This may be a common mechanism for other thiamine enzymes. The participation of a water molecule in the catalysis of the one-substrate transketolase reaction and a possible reason for the effect of the acceptor substrate on the affinity of the donor substrate for active sites are also shown.


Subject(s)
Thiamine Pyrophosphate , Transketolase , Thiamine Pyrophosphate/metabolism , Transketolase/metabolism , Thiamine/chemistry , Catalytic Domain , Catalysis , Kinetics
2.
Anal Biochem ; 613: 114022, 2021 01 15.
Article in English | MEDLINE | ID: mdl-33217405

ABSTRACT

In a recent paper, we showed the difference between the first stage of the one-substrate and the two-substrate transketolase reactions - the possibility of transfer of glycolaldehyde formed as a result of cleavage of the donor substrate from the thiazole ring of thiamine diphosphate to its aminopyrimidine ring through the tricycle formation stage, which is necessary for binding and splitting the second molecule of donor substrate [O.N. Solovjeva et al., The mechanism of a one-substrate transketolase reaction, Biosci. Rep. 40 (8) (2020) BSR20180246]. Here we show that under the action of the reducing agent a tricycle accumulates in a significant amount. Therefore, a significant decrease in the reaction rate of the one-substrate transketolase reaction compared to the two-substrate reaction is due to the stage of transferring the first glycolaldehyde molecule from the thiazole ring to the aminopyrimidine ring of thiamine diphosphate. Fragmentation of the four-carbon thiamine diphosphate derivatives showed that two glycolaldehyde molecules are bound to both coenzyme rings and the erythrulose molecule is bound to a thiazole ring. It was concluded that in the one-substrate reaction erythrulose is formed on the thiazole ring of thiamine diphosphate from two glycol aldehyde molecules linked to both thiamine diphosphate rings. The kinetic characteristics were determined for the two substrates, fructose 6-phosphate and glycolaldehyde.


Subject(s)
Transketolase/chemistry , Transketolase/metabolism , Acetaldehyde/analogs & derivatives , Acetaldehyde/chemistry , Acetaldehyde/metabolism , Biocatalysis , Borohydrides/chemistry , Coenzymes/metabolism , Fructosephosphates/chemistry , Fructosephosphates/metabolism , Kinetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Substrate Specificity , Tetroses/metabolism , Thiamine Pyrophosphate/chemistry , Thiamine Pyrophosphate/metabolism
3.
Biosci Rep ; 40(8)2020 08 28.
Article in English | MEDLINE | ID: mdl-29500317

ABSTRACT

Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.


Subject(s)
Pentosephosphates/metabolism , Pyruvates/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Tetroses/metabolism , Transketolase/metabolism , Binding Sites , Catalytic Domain , Kinetics , Molecular Dynamics Simulation , Pentosephosphates/chemistry , Protein Binding , Protein Conformation , Pyruvates/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship , Substrate Specificity , Tandem Mass Spectrometry , Tetroses/chemistry , Transketolase/chemistry
4.
Biochim Biophys Acta ; 1864(3): 280-282, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26708478

ABSTRACT

We studied the influence of the acceptor substrate of transketolase on the activity of the enzyme in the presence of reductants. Ribose-5-phosphate in the presence of cyanoborohydride decreased the transketolase catalytic activity. The inhibition is caused by the loss of catalytic function of the coenzyme-thiamine diphosphate. Similar inhibitory effect was observed in the presence of NADPH. This could indicate its possible regulatory role not only towards transketolase, but also towards the pentose phosphate pathway of carbohydrate metabolism overall, taking into account the fact that it inhibits not only transketolase but also another enzyme of the pentose phosphate pathway--glucose 6-phosphate dehydrogenase [Eggleston L.V., Krebs H.A. Regulation of the pentose phosphate cycle, Biochem. J. 138 (1974) 425-435].


Subject(s)
Pentose Phosphate Pathway , Ribosemonophosphates/chemistry , Thiamine Pyrophosphate/chemistry , Transketolase/chemistry , Borohydrides/chemistry , Carbohydrate Metabolism , Liver/chemistry , Liver/enzymology , NADP/chemistry , Reducing Agents/chemistry , Saccharomyces cerevisiae , Substrate Specificity , Thiamine Pyrophosphate/metabolism , Transketolase/antagonists & inhibitors , Transketolase/metabolism
5.
Biochim Biophys Acta ; 1844(9): 1608-18, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24929114

ABSTRACT

Studies of thiamine diphosphate-dependent enzymes appear to have commenced in 1937, with the isolation of the coenzyme of yeast pyruvate decarboxylase, which was demonstrated to be a diphosphoric ester of thiamine. For quite a long time, these studies were largely focused on enzymes decarboxylating α-keto acids, such as pyruvate decarboxylase and pyruvate dehydrogenase complexes. Transketolase, discovered independently by Racker and Horecker in 1953 (and named by Racker) [1], did not receive much attention until 1992, when crystal X-ray structure analysis of the enzyme from Saccharomyces cerevisiae was performed [2]. These data, together with the results of site-directed mutagenesis, made it possible to understand in detail the mechanism of thiamine diphosphate-dependent catalysis. Some progress was also made in studies of the functional properties of transketolase. The last review on transketolase, which was fairly complete, appeared in 1998 [3]. Therefore, the publication of this paper should not seem premature.


Subject(s)
Calcium/chemistry , Coenzymes/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Thiamine Pyrophosphate/chemistry , Transketolase/chemistry , Binding Sites , Calcium/metabolism , Coenzymes/metabolism , Glycolysis/physiology , Kinetics , Models, Molecular , Pentose Phosphate Pathway/physiology , Protein Multimerization , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/metabolism , Substrate Specificity , Thiamine Pyrophosphate/metabolism , Transketolase/metabolism
6.
Biochim Biophys Acta ; 1832(3): 387-90, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23261987

ABSTRACT

Until recently it was assumed that the transketolase-like protein (TKTL1) detected in the tumor tissue, is catalytically active mutant form of human transketolase (hTKT). Human TKT shares 61% sequence identity with TKTL1. And the two proteins are 77% homologous at the amino acid level. The major difference is the absence of 38 amino acid residues in the N-terminal region of TKTL1. Site-specific mutagenesis was used for modifying hTKT gene; the resulting construct had a 114-bp deletion corresponding to a deletion of 38 amino acid residues in hTKT protein. Wild type hTKT and mutant variant (DhTKT) were expressed in Escherichia coli and isolated using Ni-agarose affinity chromatography. We have demonstrated here that DhTKT is devoid of transketolase activity and lacks bound thiamine diphosphate (ThDP). In view of these results, it is unlikely that TKTL1 may be a ThDP-dependent protein capable of catalyzing the transketolase reaction, as hypothesized previously.


Subject(s)
Mutant Proteins/metabolism , Recombinant Proteins/metabolism , Thiamine Pyrophosphate/metabolism , Transketolase/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Kinetics , Mutagenesis, Site-Directed , Mutation , Transketolase/genetics
7.
Protein J ; 31(2): 137-40, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22210509

ABSTRACT

Catalytic activity has been demonstrated for holotransketolase in the absence of free bivalent cations in the medium. The two active centers of the enzyme are equivalent in both the catalytic activity and the affinity for the substrates. In the presence of free Ca²âº (added to the medium from an external source), this equivalence is lost: negative cooperativity is induced on binding of either xylulose 5-phosphate (donor substrate) or ribose 5-phosphate (acceptor substrate), whereupon the catalytic conversion of the bound substrates causes the interaction between the centers to become positively cooperative. Moreover, the enzyme total activity increase is observed.


Subject(s)
Calcium/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transketolase/metabolism , Calcium/analysis , Dextrans/chemistry , Holoenzymes/chemistry , Holoenzymes/isolation & purification , Holoenzymes/metabolism , Kinetics , Osmolar Concentration , Pentosephosphates/metabolism , Ribosemonophosphates/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/isolation & purification , Thiamine Pyrophosphate/analysis , Thiamine Pyrophosphate/metabolism , Transketolase/chemistry , Transketolase/isolation & purification
8.
Int J Cancer ; 124(12): 2789-96, 2009 Jun 15.
Article in English | MEDLINE | ID: mdl-19253370

ABSTRACT

Cell cycle regulation is dependent on multiple cellular and molecular events. Cell proliferation requires metabolic sources for the duplication of DNA and cell size. However, nucleotide reservoirs are not sufficient to support cell duplication and, therefore, biosynthetic pathways should be upregulated during cell cycle. Here, we reveal that glucose-6-phosphate dehydrogenase (G6PDH) and transketolase (TKT), the 2 key enzymes of oxidative and nonoxidative branches of the pentose phosphate pathway (PPP), respectively, which is necessary for nucleotide synthesis, are enhanced during cell cycle progression of the human colon cancer cell line HT29. These enhanced enzyme activities coincide with an increased ratio of pentose monophosphate to hexose monophosphate pool during late G1 and S phase, suggesting a potential role for pentose phosphates in proliferating signaling. Isotopomeric analysis distribution of nucleotide ribose synthesized from 1,2-(13)C(2)-glucose confirms the activation of the PPP during late G1 and S phase and reveals specific upregulation of the oxidative branch. Our data sustain the idea of a critical oxidative and nonoxidative balance in cancer cells, which is consistent with a late G1 metabolic check point. The distinctive modulation of these enzymes during cell cycle progression may represent a new strategy to inhibit proliferation in anticancer treatments.


Subject(s)
Adenocarcinoma/metabolism , Colonic Neoplasms/metabolism , G1 Phase/physiology , Pentose Phosphate Pathway/physiology , S Phase/physiology , Adenocarcinoma/pathology , Cell Line, Tumor , Colonic Neoplasms/pathology , Glucosephosphate Dehydrogenase/metabolism , HT29 Cells , Humans , Transketolase/metabolism
9.
Proteins ; 71(2): 541-5, 2008 May 01.
Article in English | MEDLINE | ID: mdl-18186462

ABSTRACT

The effect of the type of the cation cofactor of transketolase (i.e., Ca2+ or Mg2+) on its interaction with xylulose 5-phosphate (donor substrate) has been studied. In the presence of magnesium, the active centers of the enzyme were functionally equivalent with respect to xylulose 5-phosphate binding and exhibited identical affinities for the donor substrate. Substitution of Ca2+ for Mg2+ results in the loss of the equivalence. In particular, this becomes apparent on binding of xylulose 5-phosphates to one of the two active centers of the enzyme, which caused the second center to undergo a several fold decrease in the affinity for the donor substrate.


Subject(s)
Calcium/pharmacology , Cations, Divalent/pharmacology , Magnesium/pharmacology , Pentosephosphates/metabolism , Transketolase/metabolism , Binding Sites/drug effects , Kinetics , Saccharomyces cerevisiae/enzymology , Transketolase/drug effects
10.
Biochem Biophys Res Commun ; 361(4): 1044-7, 2007 Oct 05.
Article in English | MEDLINE | ID: mdl-17681280

ABSTRACT

The interaction of transketolase with its acceptor substrate, ribose 5-phosphate, has been studied. The active centers of the enzyme were shown to be functionally nonequivalent with respect to ribose 5-phosphate binding. Under the conditions where only one out of the two active centers of transketolase is functional, their affinities for ribose 5-phosphate are identical. The phenomenon of nonequivalence becomes apparent when the substrate interacts with one of the two active centers. As a consequence of such interaction, the affinity of the second active center for ribose 5-phosphate decreases.


Subject(s)
Ribosemonophosphates/metabolism , Transketolase/chemistry , Binding Sites , Transketolase/metabolism
11.
Bioinformatics ; 21(17): 3558-64, 2005 Sep 01.
Article in English | MEDLINE | ID: mdl-16002431

ABSTRACT

MOTIVATION: Addition of labeled substrates and the measurement of the subsequent distribution of the labels in isotopomers in reaction networks provide a unique method for assessing metabolic fluxes in whole cells. However, owing to insufficiency of information, attempts to quantify the fluxes often yield multiple possible sets of solutions that are consistent with a given experimental pattern of isotopomers. In the study of the pentose phosphate pathways, the need to consider isotope exchange reactions of transketolase (TK) and transaldolase (TA) (which in past analyses have often been ignored) magnifies this problem; but accounting for the interrelation between the fluxes known from biochemical studies and kinetic modeling solves it. The mathematical relationships between kinetic and equilibrium constants restrict the domain of estimated fluxes to the ones compatible not only with a given set of experimental data, but also with other biochemical information. METHOD: We present software that integrates kinetic modeling with isotopomer distribution analysis. It solves the ordinary differential equations for total concentrations (accounting for the kinetic mechanisms) as well as for all isotopomers in glycolysis and the pentose phosphate pathway (PPP). In the PPP the fluxes created in the TK and TA reactions are expressed through unitary rate constants. The algorithms that account for all the kinetic and equilbrium constant constraints are integrated with the previously developed algorithms, which have been further optimized. The most time-consuming calculations were programmed directly in assembly language; this gave an order of magnitude decrease in the computation time, thus allowing analysis of more complex systems. The software was developed as C-code linked to a program written in Mathematica (Wolfram Research, Champaign, IL), and also as a C++ program independent from Mathematica. RESULTS: Implementing constraints imposed by kinetic and equilibrium constants in the isotopomer distribution analysis in the data from the cancer cells eliminated estimates of fluxes that were inconsistent with the kinetic mechanisms of TK and TA. Fluxes measured experimentally in cells can be used to estimate better the kinetics of TK and TA as they operate in situ. Thus, our approach of integrating various methods for in situ flux analysis opens up the possibility of designing new types of experiments to probe metabolic interrelationships, including the incorporation of additional biochemical information. AVAILABILITY: Software is available freely at: http://www.bq.ub.es/bioqint/selivanov.htm CONTACT: martacascante@ub.edu


Subject(s)
Algorithms , Gene Expression Profiling/methods , Glucose/metabolism , Models, Biological , Pentose Phosphate Pathway/physiology , Transaldolase/metabolism , Transketolase/metabolism , Carbon Radioisotopes , Computer Simulation , Enzyme Activation , HT29 Cells , Humans , Isotope Labeling/methods , Kinetics , Multienzyme Complexes/metabolism , Software
12.
Biochem Biophys Res Commun ; 313(3): 771-4, 2004 Jan 16.
Article in English | MEDLINE | ID: mdl-14697258

ABSTRACT

Yeast transketolase, in addition to catalyzing the transferase reaction through utilization of two substrates--the donor substrate (ketose) and the acceptor substrate (aldose)--is also able to catalyze a one-substrate reaction with only aldose (glycolaldehyde) as substrate. The interaction of glycolaldehyde with holotransketolase results in formation of the transketolase reaction intermediate, dihydroxyethyl-thiamin diphosphate. Then the glycolaldehyde residue is transferred from dihydroxyethyl-thiamin diphosphate to free glycolaldehyde. As a result, the one-substrate transketolase reaction product, erythrulose, is formed. The specific activity of transketolase was found to be 0.23 U/mg and the apparent Km for glycolaldehyde was estimated as 140 mM.


Subject(s)
Acetaldehyde/analogs & derivatives , Transketolase/metabolism , Acetaldehyde/chemistry , Biochemical Phenomena , Biochemistry , Catalysis , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Kinetics , Protein Binding , Saccharomyces cerevisiae/metabolism , Time Factors , Transketolase/chemistry
13.
Biochem Biophys Res Commun ; 294(1): 155-60, 2002 May 31.
Article in English | MEDLINE | ID: mdl-12054756

ABSTRACT

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and coenzyme (thiamin diphosphate) is accompanied by the appearance of a new band, in both the absorption and CD spectra. Binding and subsequent conversion of the substrates bring about changes in this band's intensity. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of substrates during the transketolase reaction and thus to kinetically characterize its individual steps. The origin of the thiamin diphosphate induced absorption band has been postulated to be resulted from formation of a charge transfer complex or alternatively from an induced conformational transition of the enzyme. The latter brings aromatic amino acid residues into close proximity and generates the absorption. However, X-ray crystallographic and enzyme point mutation experiments cast doubts on both of these hypotheses. Here we show that the binding of thiamin diphosphate to the apotransketolase leads to the conversion of the 4'-amino tautomeric form of its aminopyrimidine ring into the N(1')H-imino tautomeric form. This imino form emerges as a result of the coenzyme's aminopyrymidine ring incorporation into the hydrophobic pocket of the transketolase active center and is stabilized through the interactions with Glu418 and Phe445 residues. The N(1')H-imino tautomeric form of thiamin diphosphate is thought to be the origin of the holotransketolase absorption band induced through the coenzyme binding.


Subject(s)
Thiamine Pyrophosphate/metabolism , Transketolase/metabolism , Catalysis , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Models, Molecular , Protein Conformation , Saccharomyces cerevisiae/enzymology , Spectrophotometry, Atomic
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