Figure 3. Irreversibility of PDHC inhibition upon preincubation with AcPH and AcPMe.

Assays were performed at 37°C in 30 mM potassium phosphate (pH 7.0) containing 3 mM NAD⁺, 0.05 mM CoA, 2 mM DTT, 0.4 mM ThDP, 2 mM MgCl₂ and indicated concentrations of pyruvate and inhibitors. A, B. Representative product accumulation curves in the presence or absence of the pyruvate P-analogs. Reactions were started by addition of 2 mM pyruvate after PDHC was preincubated for 5 min at 37°C in pyruvate-free assay medium without (grey triangles) or with (blue diamonds) the inhibitors indicated on the figures. C, D. Dependence of inhibition by AcPH or AcPMe on pyruvate added after 5 min preincubation with the inhibitors.