Thiaminepyrophosphate induced changes in the optical activity of baker's yeast transketolase

Kochetov, G. A.; Усманов, Р А; Merzlov, V.P.

doi:10.1016/0014-5793(70)80372-6

Cited by 42 publications

(18 citation statements)

References 3 publications

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“…The binding of the cofactor ThDP and the donor substrate HPA to TK can be monitored by near-UV CD spectroscopy (24,25). Holo-TK is characterized by a negative band in the CD spectrum at 320 nm.…”

Section: Measurements and Kinetics Of Hpa Binding To Tkmentioning

confidence: 99%

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Fiedler

Thorell

Sandalova

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

125

141

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Section: Measurements and Kinetics Of Hpa Binding To Tkmentioning

confidence: 99%

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Fiedler

Thorell

Sandalova

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

125

141

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“…3). Binding of thiamin diphosphate is accompanied by an increase of the band at 280 nm and the appearance of a broad negative band in the region 300-350 nm with the maximum at 320 nm (Kochetov et al, 1970;Heinrich et al, 1971). Addition of the donor substrate hydroxypyruvate leads to the inversion of the negative band (Heinrich et al, 1971;Usmanov and Kochetov, 1983).…”

Section: Circular Dichroic Measurementsmentioning

confidence: 99%

Examination of the Thiamin Diphosphate Binding Site in Yeast Transketolase by Site‐Directed Mutagenesis

Meshalkina

Nilsson

Wikner

et al. 1997

European Journal of Biochemistry

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The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis. Replacement of E l 62, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme. The two mutant enzymes show, however, significant increases in the Ko5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E l 6 2 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25 -80-fold increase in KO for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.

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“…It was also possible to follow the binding of the donor substrate directly by absorbance, but because of the impure Dxylulose 5-phosphate, rate constants were only calculated for the binding of D-fructose 6-phosphate to the wild-type of holotransketolase (Table I) (32,33). This approach has already been used for the spectroscopic characterization of the H263A mutant enzyme (18).…”

Section: Cleavage Of the Donor Substrate D-xylulose 5-phosphate In Thmentioning

confidence: 99%

Examination of Donor Substrate Conversion in Yeast Transketolase

Fiedler

Golbik

Schneider

et al. 2001

Journal of Biological Chemistry

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The cleavage of the donor substrate D-xylulose 5-phosphate by wild-type and H263A mutant yeast transketolase was studied using enzyme kinetics and circular dichroism spectroscopy. The enzymes are able to catalyze the cleavage of donor substrates, the first halfreaction, even in the absence of any acceptor substrate The pentose phosphate pathway is a major metabolic pathway in all cells. It consists of a dehydrogenase-decarboxylating system that converts D-glucose 6-phosphate to D-ribulose 5-phosphate, generating NADPHϩH ϩ for use in reductive biosynthesis, an isomerizing system that interconverts D-ribulose

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Thiaminepyrophosphate induced changes in the optical activity of baker's yeast transketolase

Cited by 42 publications

References 3 publications

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

Examination of the Thiamin Diphosphate Binding Site in Yeast Transketolase by Site‐Directed Mutagenesis

Examination of Donor Substrate Conversion in Yeast Transketolase

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