Thiamin diphosphate catalysis. Mechanistic divergence as a probe of substrate activation of pyruvate decarboxylase

Article abstract of DOI:10.1021/ja00226a044

Pyruvated decarboxylase is a thiamin diphosphate dependent enzyme that catalyzes the conversion of pyruvate to acetaldehyde and carbon dioxide. The substrate activates the enzyme, with kinetic patterns indicating a cooperative effect between two binding sites (Hill coefficient, 1.5). An alternative substrate, 3-fluoropyruvated, is converted to acetate, fluoride, and carbon dioxide. This reaction is not subject to activation, displaying normal Michaelis-Menten kinetics, but 3-fluoropyruvate activates the enzymic reaction of pyruvate. The dual reaction pattern was used as a probe of the cooperative phenomenon. Inhibition patterns show that 3-fluoropyruvate interacts with the enzyme at the same site as does pyruvate and that the affinities are similar. Since the reaction of 3-fluoropyruvate proceeds through a mechanism paralleling that of pyruvate up to the step in which carbon dioxide is lost, the step in the mechanism that is regulated occurs after the point at which the mechanisms diverge. The reaction of 3-fluoropyruvate produces enzyme-bound 2-(1-acetyl)thiamin diphosphate, which is readily hydrolyzed to produce acetate and holoenzyme, while the reaction of pyruvate produces the much less reactive enzyme-bound species 2-(1-hydroxyethyl)thiamin, which undergoes elimination of acetaldehyde to produce holoenzyme. These results suggest that the conversion of the complex of enzyme and 2-(1-hydroxyethyl)thiamin diphosphate to acetaldehyde and the holoenzyme is subject to allosteric control.