Abstract
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.
Highlights
Transketolase (TK; EC 2.2.1.1), a typical representative of the thiamine diphosphate-dependent enzymes, requires for its activity thiamine diphosphate as a coenzyme as well as divalent cations: Mg2+, Mn2+, or Ca2+ that ensure binding between the coenzyme and the apoprotein [1,2]
Its product erythrulose was determined as equimolar to the initial HPA load divided by a factor two (Table 1)
The supernatant was used for mass spectrometry
Summary
Transketolase (TK; EC 2.2.1.1), a typical representative of the thiamine diphosphate-dependent enzymes, requires for its activity thiamine diphosphate as a coenzyme as well as divalent cations: Mg2+, Mn2+, or Ca2+ that ensure binding between the coenzyme and the apoprotein [1,2]. TK is involved in the interaction between glycolysis and the pentose phosphate pathway of carbohydrate metabolism [7], which enables the cell to become adapted to different metabolic requirements. Transketolase catalyzes the reversible cleavage of the C2-C3 bond in keto sugars (donor substrates RCHOHCOCH2OH) [3,4,8]. The cleaved-off two-carbon fragment of the keto substrate (the active glycolaldehyde) becomes covalently bound to ThDP, namely (as is currently believed) to its thiazole ring and without being released into the medium, is transferred to the aldo sugar (the acceptor substrate R1CHO). The rest of the donor substrate is released into the medium as the product aldo sugar RCHO [9]
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