Cleaving of ketosubstrates by transketolase and the nature of the products formed.

The interaction of transketolase ketosubstrates with the holoenzyme has been studied. On addition of ketosubstrates cleaving both irreversibly (hydroxypyruvate) and reversibly (xylulose 5-phosphate), identical changes in the CD spectrum at 300-360 nm are observed. The changes in this spectral region, as previously shown, are due to the formation of the catalytically active holoenzyme from the apoenzyme and the coenzyme, and the cleavage of ketosubstrates by transketolase. The identity of the changes in transketolase CD spectrum caused by the addition of reversibly or irreversibly cleaving substrates indicates that in the both cases the changes are due to the formation of an intermediate product of the transketolase reaction--a glycolaldehyde residue covalently bound to the coenzyme within the holoenzyme molecule. Usually, in the course of the transferase reaction, the glycolaldehyde residue is transferred to an aldose (acceptor substrate), resulting in the recycling of the holoenzyme free of the glycolaldehyde residue. The removal of the glycolaldehyde residue from the holoenzyme appears to proceed even in the absence of an aldose. However, the glycolaldehyde cannot be found the free state because it condenses with another glycolaldehyde residue formed in the course of the cleavage of another ketosubstrate molecule yielding erythrulose.

Acceptor substrate inhibits transketolase competitively with respect to donor substrate.

Two substrates of the transketolase reaction are known to bind with the enzyme according to a ping-pong mechanism [1]. It is shown in this work that high concentrations of ribose-5-phosphate (acceptor substrate) compete with xylulose-5-phosphate (donor substrate), suppressing the transketolase activity (Ki = 3.8 mM). However, interacting with the donor-substrate binding site on the protein molecule, the acceptor substrate, unlike the donor substrate, does not cause any change in the active site of the enzyme. The data are interesting in terms of studying the regulatory mechanism of the transketolase activity and the structure of the enzyme-substrate complex.

Influence of transketolase substrates on its conformation.

Dynamics stimulation of the holotransketolase molecule revealed that the enzyme's conformation in crystal was different from that in solution. It was shown also that dissolved holotransketolase can bind aldose (the acceptor substrate) even in the absence of ketose (the donor substrate). The holotransketolase conformation did not change upon aldose binding unlike in the case of ketose binding/cleavage. Therefore the conformation of a catalytic complex of holotransketolase with an intermediate-i.e., a glycolaldehyde residue formed upon binding and subsequent cleavage of ketose-differed, at least in solution, from the conformation of both the free and aldose-complexed holotransketolase. Some structural peculiarities of the holotransketolase with the intermediate were established by means of molecular dynamics stimulation.

[Some properties of multiple forms of transketolase from baker's yeast]. / Nekotorye svoistva mnozhestvennykh form transketolazy pekarskikh drozhzhei.

It has been shown that transketolase A does not differ from the enzyme earlier described in the literature by a number of properties (lack of intersubunit disulfide bonds, identical number of sulfhydryl groups, two values of Km for thiamine pyrophosphate and identity of their absolute values). Transketolase C subunits are linked together by disulfide bonds; their total number in the enzyme molecule is five (transketolase C-1) or six (transketolase C-2). The Km values of transketolase C-1 for thiamine pyrophosphate are commensurate with those of transketolase A. Transketolase C-2 has only one Km value for thiamine pyrophosphate which is close to one of the two Km values for transketolase A. The maximal rate values for transketolases C-1 and C-2 with dihydroxyacetone as substrate differ by more than one order of magnitude.