ACTIVATION OF THIAMIN DIPHOSPHATE IN ENZYMES

Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzyme s pyruvate decarboxylase, transketolase, pyruvate dehydrogenase comple x, pyruvate oxidase, in site-specific mutant enzymes and in enzyme com plexes containing coenzyme analogues by proton/deuterium exchange dete cted by H-1-NMR spectroscopy. The respective deprotonation rate consta nt is above the catalytic constant in all enzymes investigated. The fa st deprotonation requires the presence of an activator in pyruvate dec arboxylase from yeast, showing the allosteric regulation of this enzym e to be accomplished by an increase in the C2-H dissociation rate of t he enzyme-bound thiamin diphosphate. The data of the thiamin diphospha te analogues and of the mutant enzymes show the N1' atom and the 4'-NH 2 group to be essential for the activation of the coenzyme and a conse rved glutamate involved in the proton abstraction mechanism of the enz yme-bound thiamin diphosphate. (C) 1998 Elsevier Science B.V. All righ ts reserved.