title: Activation of thiamin diphosphate in enzymes.
abstract: Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.
authors.affiliation: Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt Mothes Str. 3, D-06120 Halle, Germany. huebner@biochem.tech.uni-halle.de
authors.forename: G
authors.initials: G
authors.lastname: Hübner
authors.forename: K
authors.initials: K
authors.lastname: Tittmann
authors.forename: M
authors.initials: M
authors.lastname: Killenberg-Jabs
authors.forename: J
authors.initials: J
authors.lastname: Schäffner
authors.forename: M
authors.initials: M
authors.lastname: Spinka
authors.forename: H
authors.initials: H
authors.lastname: Neef
authors.forename: D
authors.initials: D
authors.lastname: Kern
authors.forename: G
authors.initials: G
authors.lastname: Kern
authors.forename: G
authors.initials: G
authors.lastname: Schneider
authors.forename: C
authors.initials: C
authors.lastname: Wikner
authors.forename: S
authors.initials: S
authors.lastname: Ghisla
mesh_terms: D000494:Allosteric Regulation; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology; D019906:Nuclear Magnetic Resonance, Biomolecular; D011767:Pyruvate Decarboxylase / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D011771:Pyruvate Oxidase / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D014174:Transketolase / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology
chemical_list: D011768:Pyruvate Dehydrogenase Complex; D011771:Pyruvate Oxidase; D014174:Transketolase; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate