In the complete absence of K+ and phosphate (Pi), pyruvate dehydrogenase kinase isoform 2 (PDHK2) was catalytically very active but with an elevated Km for ATP, and this activity is insensitive to effector regulation. We find that K+ or 5-fold lower levels of NH4+ markedly enhanced quenching of Trp383 fluorescence of PDHK2 by ADP and ATP. K+ binding caused an -40-fold decrease in the equilibrium dissociation constants (Kd) for ATP from -120 to 3.0 μM and an ∼25-fold decrease in Kd for ADP from ∼950 to 38 μM. Linked reductions in Kd of PDHK2 for K+ were from ∼30 to ∼0.75 mM with ATP bound and from ∼40 to ∼1.7 mM with ADP bound. Without K+, there was little effect of ADP on pyruvate binding, but with 100 mM K+ and 100 μM ADP, the L0.5 of PDHK2 for pyruvate was reduced by ∼14 fold. In the absence of K+, Pi had small effects on ligand binding. With 100 mM K+, 20 mM Pi modestly enhanced binding of ADP and hindered pyruvate binding but markedly enhanced the binding of pyruvate with ADP; the L0.5 for pyruvate was specifically decreased ∼125-fold with 100 μM ADP. Pi effects were minimal when NH4 + replaced K+. We have quantified coupled binding of K+ with ATP and ADP and elucidated how linked K+ and Pi binding are required for the potent inhibition of PDHK2 by ADP and pyruvate.
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