Fig. 3. .

Performance of known inhibitors in the ODC-PEPC-MDH and PEPC-MDH assays ( = 60% Infinity CO₂, ▴ = 45% Infinity CO₂, ▾ = 30% Infinity CO₂, ♦ = 15% Infinity CO₂). Data were collected under optimized screening conditions as described unless otherwise noted. Compounds were allowed to equilibrate with enzymes for 20 min prior to addition of substrate. Percent inhibition is defined as (1 − v_i/v_o) × 100. At Infinity, CO₂ percentages lower than 45% reaction rates were calculated from the first 10 min of the reaction rather than the normal 20 min used at higher percentages due to limitations in assay linear time (see Fig. 1D). For PEPC-MDH assay results, rates were determined from the first 5 min (see Fig. 2B). (A) Performance of difluoromethylornithine (DFMO), a known ornithine decarboxylase (ODC) inhibitor, in the ODC-PEPC-MDH assay with varying amounts of linking enzymes. Note that linking enzyme concentrations do not effect the performance of DFMO. (B) Performance of baicalein, a known inhibitor of PEPC, at several different final linking mix concentrations in the ODC-PEPC-MDH assay. At normal screening conditions (60% Infinity CO₂) no inhibition is seen, even from this submicromolar inhibitor. However, upon dilution of the linking mix, inhibition becomes apparent. (C) Performance of isoquinoline, a known inhibitor of MDH in the ODC-PEPC-MDH assay at several Infinity CO₂ percentages. No inhibition was seen in any condition tested. Reaction rates at Infinity CO₂ concentrations lower than 45% were calculated from the first 10 min of data due to limitations in assay linear time (see Fig. 1D). (D) Performance of DFMO in the PEPC-MDH assay system at varying levels of Infinity CO₂. (E) Performance of baicalein in the PEPC-MDH assay system at varying levels of Infinity CO₂. As with the ODC-PEPC-MDH system, a dependency on the amount of linking enzymes present greatly effects the amount of inhibition seen. (F) Performance of isoquinoline in the PEPC-MDH assay. No inhibition was observed under any conditions tested. *Literature value,²⁶ **Literature value,²⁷ ***Literature value.²⁸