Studies on the Reconstitution of Apotransketolase with Thiamine Pyrophosphate and Analogs of the Coenzyme

Abstract

The binding of thiátmine pyrophosphate to apotransketolase from baker's yeast has been studied by measuring the cofactor‐dependent activity after passage through a column of Sephadex G‐25 or dialysis, respectively. At pH values >6.5, the cofactors Mg2+ and thiamine pyrophosphate were lost upon gel filtration or dialysis, indicating reversible binding. Thiamine pyrophosphate alone was able to activate and partially reconstitute transketolase activity in the absence of divalent cations. Using activity and fluorescence‐quenching measurements the reconstitution process of transketolase was studied as a function of different parameters such as pH, temperature, ionic strength, donor and acceptor substrates and various divalent cations. The rate of recombination of transketolase from its components, apotransketolase, thiamine pyrophosphate and divalent cations, increases in the order Ni < Mg < Co < Mn < Ca, whereas the catalytic activity of the enzyme does not depend on the nature of the complexing metal ion. Extensively dialyzed apotransketolase is activated by SO2−4 ions in 10—20 mM concentration. The binding constants for thiamine pyrophosphate determined by activity measurements were 4.8 μM in the absence and 1.0 μM in the presence of 4.4 mM Mg2+. A Km of 1.1 μM (5 mM Mg2+) was obtained from fluorescence quenching experiments. A Km of 5.5 μM was obtained for 2′‐ethylthiamine pyrophosphate. The V for the latter compound was 27% of that of thiamine pyrophosphate. For the following competitive inhibitors of thiamine pyrophosphate the inhibitor constants (Ki) were determined: oxythiamine pyrophosphate, 0.03 μM; tetrahydrothiamine pyrophosphate, 0.40 μM; thiochrome pyrophosphate, 6.3 μM; pyrithiamine pyrophosphate, 110 μM; inorganic pyrophosphate, 4200 μM. The relevance of the charge transfer interaction between thiamine pyrophosphate and apotransketolase is discussed. Copyright © 1972, Wiley Blackwell. All rights reserved