Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.

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.

Thiamine is a vitamin essential for the function of central metabolic enzymes, of which pyruvate dehydrogenase (PDH) possesses one of the broadest regulations. Diurnal effects of thiamine supplementation on energy metabolism have previously been shown for the rat brain. Here, we report data on the diurnal changes and the effects of thiamine administration to rats on the function of thiamine-dependent enzymes in the cerebral cortex, heart, and liver. The most pronounced diurnal differences were found at the level of cerebral PDH activity. Analysis of PDH phosphorylation in five rat tissues revealed diurnal and thiamine-dependent differences in the cerebral cortex and heart. The expression of tissue-specific PDH kinases PDK3 and PDK4 showed a daytime-dependent response to thiamine administration in the cerebral cortex and heart, respectively. In addition, cardiac PDK4 expression was doubled in the evening, compared to morning. Furthermore, cerebral cortex demonstrated tissue-specific diurnal changes in thiamine diphosphate (ThDP) and monophosphate levels. Elevation of blood, cardiac, and cerebral ThDP was more effective upon the evening thiamine administration. Importantly, only ThDP was elevated in the rat cerebral cortex exclusively after evening thiamine supplementation. Coenzyme role of ThDP and/or other thiamine functions in nerve tissue reduced the existing daytime changes in animal behavior and ECG parameters. The reported data on diurnal regulation of central energy metabolism as well as the diurnal difference in thiamine accumulation in the cerebral cortex, heart, and other tissues are of clinical importance, as high doses of thiamine are used for the treatment of acute thiamine deficiencies and many other mostly neurological diseases in patients.