The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

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Iatrogenic tracheal rupture is a dangerous complication with potentially high postoperative mortality, mostly influenced by the underlying disease. Early surgical repair must be the preferred treatment.

Patients with an R1 situation have a survival rate of 14% comparable to curative resected patients (RO) in stage III. Adjuvant radiation had no clear effect on survival. Patients with macroscopic tumor (R2) should receive palliative treatment after the operation depending on their condition.

Abstract. Activation. of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of. thiam~n , 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 NI' atom and the '4'-NH 2 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.