Determination of the binding constant of thiamine diphosphate in transketolase from baker's yeast by circular dichroism titration

Heinrich, C.P.; Schmidt, Dieter

doi:10.1007/bf01935085

Cited by 12 publications

(7 citation statements)

References 4 publications

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“…The proposed mechanism explains various data reporting the coenzyme's affinity to apoTK in the presence of magnesium ions in the literature [5–7,25]. Some authors report a negative cooperativity, albeit slightly pronounced [5], while others call into question the nonequivalency of the enzyme's active centers on ThDP binding [6,7]. All of these data were received in the absence of the donor substrate and were correlated with the results obtained.…”

Section: Discussionsupporting

confidence: 77%

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Donor substrate regulation of transketolase

Esakova

Meshalkina

Golbik

et al. 2004

European Journal of Biochemistry

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The influence of substrates on the interaction of apotransketolase with thiamin diphosphate was investigated in the presence of magnesium ions. It was shown that the donor substrates, but not the acceptor substrates, enhance the affinity of the coenzyme either to only one active center of transketolase or to both active centers, but to different degrees in each, resulting in a negative cooperativity for coenzyme binding. In the absence of donor substrate, negative cooperativity is not observed. The donor substrate did not affect the interaction of the apoenzyme with the inactive coenzyme analogue, N3¢-pyridyl-thiamin diphosphate. The influence of the donor substrate on the coenzymeapotransketolase interaction was predicted as a result of formation of the transketolase reaction intermediate 2-(a,b-dihydroxyethyl)-thiamin diphosphate, which exhibited a higher affinity to the enzyme than thiamin diphosphate 1. The enhancement of thiamin diphosphate's affinity to apotransketolase in the presence of donor substrate is probably one of the mechanisms underlying the substrate-affected transketolase regulation at low coenzyme concentrations.

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Section: Discussionsupporting

confidence: 77%

“…However, contrasting data have been published regarding the affinity of the coenzyme to the apoenzyme of TK (apoTK) in the presence of magnesium ions. Some authors report a negative cooperativity, albeit slightly pronounced [5], while others call into question the nonequivalency of the enzyme's active centers on ThDP binding [6,7].…”

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confidence: 99%

Donor substrate regulation of transketolase

Esakova

Meshalkina

Golbik

et al. 2004

European Journal of Biochemistry

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“…When Mg 2+ is used as a cofactor instead of Ca 2+ , the two active centers exhibit the same affinity for TDP (18, 19) or differ insignificantly in this respect (6, 15, 20). The presence of X5P (a donor substrate), however, causes one center to increase its affinity for TDP considerably (compare 1 and 2 in Fig.…”

Section: Tdp Binding By Apotk In the Presence Of Mg2+mentioning

confidence: 99%

Functional nonequivalence of transketolase active centers

Kochetov

Sevostyanova

2010

IUBMB Life

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SummaryTransketolase (TK, EC 2.2.1.1), the key enzyme of the nonoxidative branch of pentose phosphate pathway of hydrocarbon transformation, plays an important role in a system of substrate rearrangement between pentose shunt and glycolysis, acting as a reversible link between the two metabolic pathways. In addition, it supplies precursors for biosyntheses of nucleotides, aromatic amino acids, and vitamins. In plants, the enzyme plays a central role in the Calvin cycle. TK catalyzes interconversion of sugar phosphates. Thiamine diphosphate (TDP) and bivalent cations serve as its cofactors. Being a typical TDP-dependent enzyme, TK is the least complex representative of this group of enzymes, and this accounts for its use as a model in studies of their structure and mechanism of action. TK is readily crystallized, this being the reason why the first crystal X-ray structure analysis of TDP-dependent enzymes was performed with a TK sample. Both the general structure of TK and the structures of its active centers have been studied in detail. In this article, we review experimental evidence of functional nonequivalence of the two active centers of TK, which are known to be identical by crystal X-ray structure analysis. half-of-the-sites reactivity.Abbreviations TK, Transketolase; TDP, thiamine diphosphate; X5P, xylulose 5-phosphate; R5P, ribose 5-phosphate; F6P, fructose 6-phosphate; DHETDP, dihydroxyethylthiamine diphosphate.

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“…In the presence of Ca 2+ , there is negative coopera tivity in the binding of TDP by the active centers of TK [5 9]. In the presence of Mg 2+ , the nonequivalence in behavior of the active centers with respect to TDP is either absent [10,11] or poorly manifested [9]. X Ray analysis fails to detect the nonequivalence of the active centers [3,4,12] that is observed on the functional level.…”

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confidence: 95%

Effect of Transketolase Substrates on Holoenzyme Reconstitution and Stability

Esakova

Khanova

Meshalkina

et al. 2005

Biochemistry (Moscow)

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The influence of transketolase substrates on the interaction of apotransketolase with its coenzyme thiamine diphosphate (TDP) and on the stability of the reconstituted holoenzyme was studied. Donor substrates increased the affinity of the coenzyme for transketolase, whereas acceptor substrate did not. In the presence of magnesium ions, the active centers of transketolase initially identical in TDP binding lose their equivalence in the presence of donor substrates. The stability of transketolase depended on the cation type used during its reconstitution--the holoenzyme reconstituted in the presence of calcium ions was more stable than the holoenzyme produced in the presence of magnesium ions. In the presence of donor substrate, the holoenzyme stability increased without depending on the cation used during the reconstitution. Donor substrate did not influence the interaction of apotransketolase with the inactive analog of the coenzyme N3'-pyridyl thiamine diphosphate and did not stabilize the transketolase complex with this analog. The findings suggest that the effect of the substrate on the interaction of the coenzyme with apotransketolase and on stability of the reconstituted holoenzyme is caused by generation of 2-(alpha,beta-dihydroxyethyl)thiamine diphosphate (an intermediate product of the transketolase reaction), which has higher affinity for apotransketolase than TDP.

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Determination of the binding constant of thiamine diphosphate in transketolase from baker's yeast by circular dichroism titration

Cited by 12 publications

References 4 publications

Donor substrate regulation of transketolase

Donor substrate regulation of transketolase

Functional nonequivalence of transketolase active centers

Effect of Transketolase Substrates on Holoenzyme Reconstitution and Stability

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