Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyses the one-substrate reaction that involves only ketose. The mechanism of this reaction was unknown.  Here we show that hydroxypyruvate  serves as a substrate for one-substrate reaction and the reaction product is erythrulose rather than glycol aldehyde, as was shown earlier for xylulose 5-phosphate. 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 from the first ketose molecule (the product of the first half reaction) remains bound to the enzyme and is not released into the medium, waiting for condensation with the second molecule of glycol aldehyde, the product of the half reaction with the second molecule of the substrate. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate  results in the remainder of the active glycol aldehyde being equiprobably bound to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We show also, that after the first formed molecule of glycolaldehyde passes from the thiazole ring of thiamine diphosphate to its aminopyrimidine ring, binding and splitting of the second ketosubstrate molecule occurs on the thiazole ring. The condensation of two molecules of glycolaldehyde occurs on the thiazole ring of thiamine diphosphate.

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