One-Substrate Transketolase-Catalyzed Reaction

Bykova, Irina; Solovjeva, Olga N.; Meshalkina, L. E.; Kovina, Marina V.; Kochetov, G. A.

doi:10.1006/bbrc.2000.4199

Cited by 39 publications

(23 citation statements)

References 12 publications

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“…devoid of acceptor R5P). It could be demonstrated for TKTs from other organisms that X5P is slowly converted to G3P and erythrulose (carboligation product of two glycolaldehyde molecules) in the absence of an acceptor (31).…”

Section: Methodsmentioning

confidence: 99%

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

Mitschke

Parthier

Schröder-Tittmann

et al. 2010

Journal of Biological Chemistry

169

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The crystal structure of human transketolase (TKT), a thiamine diphosphate (ThDP) and Ca 2؉ -dependent enzyme that catalyzes the interketol transfer between ketoses and aldoses as part of the pentose phosphate pathway, has been determined to 1.75 Å resolution. The recombinantly produced protein crystallized in space group C2 containing one monomer in the asymmetric unit. Two monomers form the homodimeric biological assembly with two identical active sites at the dimer interface. Transketolase (TKT 3 ; EC 2.2.1.1) is a ubiquitous enzyme in cellular carbon metabolism and requires thiamine diphosphate (ThDP), the biologically active derivative of vitamin B1, and Ca 2ϩ ions as cofactors for enzymatic activity (1, 2). TKT catalyzes the reversible transfer of two-carbon (1,2-dihydroxyethyl) units from ketose phosphates to the C1 position of aldose phosphates and thus provides, together with the Schiff base-forming transaldolase, a reversible link between glycolysis and the pentose phosphate pathway. This shunt permits cells a flexible adaptation to different metabolic needs as the pentose phosphate pathway supplies intermediates for other metabolic pathways; generates precursors for biosynthesis of nucleotides, aromatic amino acids, and vitamins; and further produces NADPH for sustaining the glutathione level and for reductive biosynthetic pathways of, for example, cholesterol and fatty acids.TKT acts on different ketose phosphate (donor) and aldose phosphate (acceptor) substrates of variable carbon chain length (3-7 carbons) in two major, essentially reversible reactions. REACTIONS 1 AND 2A simplified reaction scheme for the TKT-catalyzed conversion of substrates X5P and R5P into products S7P and G3P is shown in Fig. 1. The reaction cycle can be subdivided into a donor half-reaction (donor ligation and cleavage) and an acceptor half-reaction (acceptor ligation and product liberation) (3).After formation of the reactive ylide form of ThDP, the C2 carbanion of ThDP attacks the carbonyl of donor X5P in a nucleophilic manner to yield the covalent donor-ThDP adduct X5P-ThDP (step 1). Ionization of C3-OH and cleavage of the scissile C2-C3 bond of X5P-ThDP results in the formation of product G3P and of the 1,2-dihydroxylethyl-ThDP (DHEThDP) carbanion/enamine intermediate (step 2). This intermediate may then react with either G3P (reverse reaction of step 2) or R5P in competing equilibria. In the latter case, C2␣ of DHE-ThDP ligates to C1 of R5P (in the acyclic form), yielding * This work was supported by a grant from the "Fonds der Chemischen Industrie" (stipend to S. L.) and the Deutsche Forschungsgemeinschaft-funded Gö ttingen Graduate School for Neurosciences and Molecular Biosciences (to K. T.

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Section: Methodsmentioning

confidence: 99%

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

Mitschke

Parthier

Schröder-Tittmann

et al. 2010

Journal of Biological Chemistry

169

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“…d ‐ribose‐5‐phosphate was measured by kinetic assay of its reaction with 35 μM d ‐xylulose‐5‐phosphate catalyzed by transketolase in 0.6‐ml mixtures containing 25 mM glycylglycine, pH 7.3, 5 mM MgCl 2 , 0.22 mM thiamine pyrophosphate, 5 mM sodium arsenate, 1 mM NAD, 0.1 mg/ml bovine serum albumin, 5 μg/ml transketolase (dissolved in 250 mM glycylglycine, pH 7.3) and 60 m‐unit d ‐glyceraldehyde‐3‐phosphate dehydrogenase (pre‐assayed under these conditions with 80 μM d ‐glyceraldehyde‐3‐phosphate as substrate). Transketolase forms one mole of d ‐glyceraldehyde‐3‐phosphate per mole of d ‐xylulose‐5‐phosphate either by monosubstrate reaction or by bisubstrate reaction with d ‐ribose‐5‐phosphate [22,23]. The formation of d ‐glyceraldehyde‐3‐phosphate is coupled to its arsenate and NAD‐dependent oxidation catalyzed irreversibly by d ‐glyceraldehyde‐3‐phosphate dehydrogenase.…”

Section: Methodsmentioning

confidence: 99%

“…The increase of A 265 caused by the enzyme itself was determined and used to calculate the true decrease due to AMP deamination. [22,23]. The formation of D-glyceraldehyde-3-phosphate is coupled to its arsenate and NAD-dependent oxidation catalyzed irreversibly by D-glyceraldehyde-3-phosphate dehydrogenase.…”

Section: Hplcmentioning

confidence: 99%

Hydrolysis of the phosphoanhydride linkage of cyclic ADP‐ribose by the Mn²⁺‐dependent ADP‐ribose/CDP‐alcohol pyrophosphatase

et al. 2009

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a b s t r a c tCyclic ADP-ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP-ribose, form and hydrolyze the N 1 -glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) and found that cADPR is an ADPRibase-Mn ligand and substrate. ADPRibase-Mn activity on cADPR was 65-fold less efficient than on ADP-ribose, the best substrate. This is similar to the ADP-ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase-Mn was N 1 -(5-phosphoribosyl)-AMP, suggesting a novel route for cADPR turnover.

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“…The X-ray analysis of the TK-reaction intermediate, DHEThDP, in the TK active center suggests that His103 directly participates in the stabilization of the intermediate by forming a hydrogen bond with the oxygen of its b-hydroxyl group [2]. Note, that the rate of one-substrate reaction is limited by the rate of glycolaldehyde release from DHEThDP [14,15] (i.e., by the stability of the ''active glycolaldehyde'' intermediate formed during binding and splitting of the donor substrate).…”

Section: Interaction Of H103a Mutant Apotk With Thdpmentioning

confidence: 99%

“…TK, being a typical transferase, is able to catalyze the socalled one-substrate reaction where only the donor substrate is utilized in the absence of the acceptor substrate [14][15][16]. As for the two-substrate reaction, the formation of the intermediate product, a,b-dihydroxyethyl-thiamin diphosphate (DHEThDP), (i.e., glycolaldehyde attached to ThDP, the so-called ''active glycolaldehyde'') is registered.…”

Section: Introductionmentioning

confidence: 99%

Kinetic study of the H103A mutant yeast transketolase

et al. 2004

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Data from site-directed mutagenesis and X-ray crystallography show that His103 of holotransketolase (holoTK) does not come into contact with thiamin diphosphate (ThDP) but stabilizes the transketolase (TK) reaction intermediate, ,-dihydroxyethyl-thiamin diphosphate, by forming a hydrogen bond with the oxygen of its -hydroxyethyl group [Eur. J. Biochem. 233 (1995) 750; Proc. Natl. Acad. Sci. USA 99 (2002) 591]. We studied the influence of His103 mutation on ThDP-binding and enzymatic activity. It was found that mutation does not affect the affinity of the coenzyme to apotransketolase (apoTK) in the presence of Ca 2þ (a cation found in the native holoenzyme) but changes all the kinetic parameters of the ThDP-apoTK interaction in the presence of Mg 2þ (a cation commonly used in ThDP-dependent enzymes studies). It was concluded that the structures of TK active centers formed in the presence of Mg 2þ and Ca 2þ are not identical. Mutation of His103 led to a significant acceleration of the one-substrate reaction but a slow down of the two-substrate reaction so that the rates of both types of catalysis became equal. Our results provide evidence for the intermediate-stabilizing function of His103.

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One-Substrate Transketolase-Catalyzed Reaction

Cited by 39 publications

References 12 publications

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

Hydrolysis of the phosphoanhydride linkage of cyclic ADP‐ribose by the Mn²⁺‐dependent ADP‐ribose/CDP‐alcohol pyrophosphatase

Kinetic study of the H103A mutant yeast transketolase

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One-Substrate Transketolase-Catalyzed Reaction

Cited by 39 publications

References 12 publications

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

Hydrolysis of the phosphoanhydride linkage of cyclic ADP‐ribose by the Mn2+‐dependent ADP‐ribose/CDP‐alcohol pyrophosphatase

Kinetic study of the H103A mutant yeast transketolase

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Hydrolysis of the phosphoanhydride linkage of cyclic ADP‐ribose by the Mn²⁺‐dependent ADP‐ribose/CDP‐alcohol pyrophosphatase