Which stage of the process of apotransketolase interaction with thiamine diphosphate is affected by the regulatory activity of the donor substrate?

The interaction of thiamine diphosphate (ThDP) with transketolase (TK) involves at least two stages: TK + ThDP⇆^Kd TK⋯ThDP _k-1⇆^k+1TK*-ThDP During the first stage, an inactive intermediate complex (TK⋯ThDP) is formed, which is then transformed into a catalytically active holoenzyme (TK* - ThDP). The second stage is related to conformational changes of the protein. In the preceding publication (Esakova, O. A., Meshalkina, L. E., Golbik, R., Hübner, G., and Kochetov, G. A. Eur. J. Biochem. 2004, 271, 4189 - 4194) we reported that the affinity of ThDP for TK considerably increases in the presence of the donor substrate, which may be a mechanism whereby the activity of the enzyme is regulated under the conditions of the coenzyme deficiency. Here, we demonstrate that the substrate affects the stage of the reverse conformational transition, characterized by the constant k-1: in the presence of the substrate, its value is decreased several fold, whereas K_d and k₊₁ remain unchanged.