The number of active sites in a molecule of transketolase

Kochetov, G. A.; Meshalkina, L. E.; Усманов, Р А

doi:10.1016/0006-291x(76)90450-2

Cited by 35 publications

(20 citation statements)

References 8 publications

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“…TK from Saccharomyces cerevisiae is composed from two identical subunits with a molecular mass of 74.2 kDa per monomer and it has two active centers [4][5][6]. TK was the first ThDP-dependent enzyme to have its crystal structure solved.…”

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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Section: Introductionmentioning

confidence: 99%

Kinetic study of the H103A mutant yeast transketolase

et al. 2004

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“…did not affect fit quality). The remaining part of the curve depends only on K 3 , since the first site is already occupied. The best-fit value of K?…”

Section: Resultsmentioning

confidence: 99%

“…TDP binding to apoTK results in an absorption maximum at 315-320 nm [8][9][10][11], whose intensity is proportional to the amount of the reconstituted holoenzyme [12][13][14], The kinetics of the absorbance increase was recorded in an Aminco DW-2000 spectrophotometer operated in a two-wavelength mode (the reference wavelength was 360 nm) until the equilibrium level was attained (usually 10 min). The reconstitution was carried out in 10 mM Tris-HCl buffer, pH 7.35, containing 2 mM CaCl 2 .…”

Section: Kinetic Measurementsmentioning

confidence: 99%

Kinetic mechanism of active site non‐equivalence in transketolase

Kovina¹,

Selivanov²,

Kochevova³

et al. 1997

FEBS Letters

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The two-step mechanism of coenzyme (TDP) binding to apotransketolase has been examined by kinetic modeling, and the rate and equilibrium constants for each binding step for two active sites have been determined. The dissociation constants for the primary fast binding step and the forward rate constants for the secondary slow binding step have been shown to be similar for two active sites. The backward rate constants for the secondary binding step are different for two active sites, providing the kinetic mechanism of their non-equivalence in TDP binding.

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“…The binding of ThDP to apoTK and the formation of a catalytically active holoenzyme (TK*-ThDP in Scheme 1) is accompanied by the appearance of a new absorption band (in the 290 -340 nm range) (18,19), the intensity of which is strictly correlated with the amount of coenzyme bound in the active centers of TK (19,20). This approach was used to determine the affinity of the coenzyme to TK (5,19) and to study the interaction of ThDP to the apoenzyme.…”

Section: Stopped-flow Kinetics Of the Thdp Bindingmentioning

confidence: 99%

Which stage of the process of apotransketolase interaction with thiamine diphosphate is affected by the regulatory activity of the donor substrate?

et al. 2007

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SummaryThe interaction of thiamine diphosphate (ThDP) with transketolase (TK) involves at least two stages:During the first stage, an inactive intermediate complex (TKÁÁÁThDP) is formed, which is then transformed into a catalytically active holoenzyme (TK* -ThDP). The second stage is related to conformational changes of the protein. In the preceding publication (Esakova, O. A., Meshalkina, L. E., Golbik, R., Hu¨bner, G., and Kochetov, G. A. Eur. J. Biochem. 2004, 271, 4189 -4194) we reported that the affinity of ThDP for TK considerably increases in the presence of the donor substrate, which may be a mechanism whereby the activity of the enzyme is regulated under the conditions of the coenzyme deficiency. Here, we demonstrate that the substrate affects the stage of the reverse conformational transition, characterized by the constant k 71 : in the presence of the substrate, its value is decreased several fold, whereas K d and k þ1 remain unchanged.

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The number of active sites in a molecule of transketolase

Cited by 35 publications

References 8 publications

Kinetic study of the H103A mutant yeast transketolase

Kinetic study of the H103A mutant yeast transketolase

Kinetic mechanism of active site non‐equivalence in transketolase

Which stage of the process of apotransketolase interaction with thiamine diphosphate is affected by the regulatory activity of the donor substrate?

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