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

Abstract

SummaryThe interaction of thiamine diphosphate (ThDP) with transketolase (TK) involves at least two stages:During the first stage, an inactive intermediate complex (TKÁÁÁThDP) 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., Hu¨bner, 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 71 : in the presence of the substrate, its value is decreased several fold, whereas K d and k þ1 remain unchanged.

C−C Bond-Forming Lyases in Organic Synthesis

“…10 Although the activation mechanism of ThDP-dependent enzymes is well-known, 162,171 and the enamine character of the key intermediate was proven spectroscopically, 172 detailed research on the topic is in progress. 164,165,[173][174][175][176][177][178][179][180] Transketolase was identified for the first time by Racker et al, 181,182 in S. cerevisiae, and subsequently found in a wide variety of sources such as other yeasts, 183,184 spinach, [185][186][187] wheat leaves, 186 and also mammalian cells, such as rat liver cells, 188 or human leukocytes. 189 TK from E. coli was also cloned and overexpressed in recombinant E. coli strains, and it is easily available for preparative scale reactions.…”

“…Although the activation mechanism of ThDP-dependent enzymes is well-known, , and the enamine character of the key intermediate was proven spectroscopically, detailed research on the topic is in progress. ,,− …”

The uremic toxin oxythiamine causes functional thiamine deficiency in end-stage renal disease by inhibiting transketolase activity

“…40 This may have contributed to discordant reports of decreased transketolase activity in ESRD patients. Studies by Lonergan and co-workers 2, 3 and Kopple et al 41 used a modified method of transketolase activity assessment developed by Dreyfus 42 which was subsequently found to lack specificity and reproducibility [43][44][45] and is no longer used.…”

“…When transketolase is in the presence of substrate, exchange of TPP cofactor and OTPP inhibitor is very slow. 40 The absolute level of transketolase activity relates to metabolic function in the pentosephosphate pathway whereas αETKA captures the competitive inhibition of transketolase in ESRD patients and unsaturation of transketolase by TPP in thiamine deficiency.…”