HOW THIAMINE DIPHOSPHATE IS ACTIVATED IN ENZYMES

The controversial question of how thiamine diphosphate, the biological ly active form of vitamin B-1, is activated in different enzymes has b een addressed. Activation of the coenzyme was studied by measuring the rmodynamics and kinetics of deprotonation at the carbon in the 2-posit ion (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'-positi on in the pyrimidine ring activated the 4'-amino group to act as an ef ficient proton acceptor for the C2 proton. The protein component accel erated the deprotonation of the C2 atom by several orders of magnitude , beyond the rate of the overall enzyme reaction. Therefore, the earli er proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.