AG-348 activates a spectrum of recombinant mtPK-R enzymes. (A) PK-R tetramer with sites of mtPK-R enzymes tested in Figure 3B highlighted in red. For clarity, each mutation site is only shown in a single monomer. (B) Fold activation and AC50 values of AG-348 treatment of mtPK-R enzymes (AG-348 = 10 μM, PEP, as listed in the supplemental table). (C) Activity of recombinant R532W mtPK-R enzyme stimulated with PEP with or without preincubation with AG-348 (AG-348 = 5 μM). (D) Activity of recombinant R532W mtPK-R enzyme incubated with indicated concentration of FBP or AG-348 (PEP = 0.05 mM). Panels C and D show the average of 3 technical replicates. (E) Residual activity over time of WT or R510Q recombinant enzymes following incubation at 53°C (AG-348 = 5 μM, PEP = 2 mM). (F) Off-rate measurement of AG-348 or FBP (both at 5 μM final assay concentration, PEP = 2 mM) from recombinant R510Q enzyme. All error bars are standard deviations.
