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Background: Thiamine diphosphate (ThDP), an indispensable cofactor for oxidative energy metabolism, is synthesized through the reaction thiamine + ATP ⇆ ThDP + AMP, catalyzed by thiamine pyrophosphokinase 1 (TPK1), a cytosolic dimeric enzyme. It was claimed that the equilibrium of the reaction is in favor of the formation of thiamine and ATP, at odds with thermodynamic calculations. Here we show that this discrepancy is due to feedback inhibition by the product ThDP., Methods: We used a purified recombinant mouse TPK1 to study reaction kinetics in the forward (physiological) and for the first time also in the reverse direction., Results: K eq values reported previously are strongly underestimated, due to the fact the reaction in the forward direction rapidly slows down and reaches a pseudo-equilibrium as ThDP accumulates. We found that ThDP is a potent non-competitive inhibitor (K i  ≈ 0.4 μM) of the forward reaction. In the reverse direction, a true equilibrium is reached with a K eq of about 2 × 10 -5 , strongly in favor of ThDP formation. In the reverse direction, we found a very low K m for ThDP (0.05 μM), in agreement with a tight binding of ThDP to the enzyme., General Significance: Inhibition of TPK1 by ThDP explains why intracellular ThDP levels remain low after administration of even very high doses of thiamine. Understanding the consequences of this feedback inhibition is essential for developing reliable methods for measuring TPK activity in tissue extracts and for optimizing the therapeutic use of thiamine and its prodrugs with higher bioavailability under pathological conditions., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)