The binding of thiltmine 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 Mga+ 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 SO?-ions in 10-20 mM concentration. The binding constants for thiamine pyrophosphate determined by activity measurements were 4.8 pM in the absence and 1.0 pM in the presence of 4.4 mM Mg2+. A K , of 1.1 pM (5 mM Mg2+) was obtained from fluorescence quenching experiments. A K , of 5.5 p.M was obtained for 2'-ethylthiamine pyrophosphate. The V for the latter compound was 27O/, of that of thiamine pyrophosphate. For the following competitive inhibitors of thiamine pyrophosphate the inhibitor constants (Ki) were determined : oxythiamine pyrophosphate, 0.03 p.M ; tetrahydrothiamine pyrophosphate, 0.40 pM; thiochrome pyrophosphate, 6.3 pM; pyrithiamine pyrophosphate, 110 pM ; inorganic pyrophosphate, 4200 pM.The relevance of the charge transfer interaction between thiamine pyrophosphate and apotransketolase is discussed.Transketolase from baker's yeast requires the addition of thiamine pyrophosphate and metal ions (Me2+) for activity. Considerable progress has been made in elucidating its mechanism of action. As shown by Holzer and co-workers, transketolase reactions proceed via "active glycolaldehyde" as intermediate [I]. Up to now, only a few experiments have been carried out in order to obtain a better understanding of the nature of the binding of thiamine pyrophosphate to transketolase [2,3]. The present paper reports on the reversibility of coenzyme binding to apotransketolase and on the influence of parameters such as temperature, pH, ionic strength, divalent cations and substrates on the reconstitution of transketolase from baker's Abbreviation. CD, circular dichroism. Enzymes. Transketolase or sedoheptulose-7-phosphate : ~-glyceraldehyde-3-phosphate glycolaldehyde transferase (EC 2.2
