New Role of Water in Transketolase Catalysis.

Abstract

Transketolase catalyzes the interconversion of keto and aldo sugars. Its coenzyme is thiamine diphosphate. The binding of keto sugar with thiamine diphosphate is possible only after C2 deprotonation of its thiazole ring. It is believed that deprotonation occurs due to the direct transfer of a proton to the amino group of its aminopyrimidine ring. Using mass spectrometry, it is shown that a water molecule is directly involved in the deprotonation process. After the binding of thiamine diphosphate with transketolase and its subsequent cleavage, a thiamine diphosphate molecule is formed with a mass increased by one oxygen molecule. After fragmentation, a thiamine diphosphate molecule is formed with a mass reduced by one and two hydrogen atoms, that is, HO and H2O are split off. Based on these data, it is assumed that after the formation of holotransketolase, water is covalently bound to thiamine diphosphate, and carbanion is formed as a result of its elimination. This may be a common mechanism for other thiamine enzymes. The participation of a water molecule in the catalysis of the one-substrate transketolase reaction and a possible reason for the effect of the acceptor substrate on the affinity of the donor substrate for active sites are also shown.