Critical role of specific ions for ligand-induced changes regulating pyruvate dehydrogenase kinase isoform 2

In the complete absence of K⁺ and phosphate (P_i), pyruvate dehydrogenase kinase isoform 2 (PDHK2) was catalytically very active but with an elevated K_m 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 (K_d) for ATP from ^-120 to 3.0 μM and an ∼25-fold decrease in K_d for ADP from ∼950 to 38 μM. Linked reductions in K_d 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 L_0.5 of PDHK2 for pyruvate was reduced by ∼14 fold. In the absence of K⁺, P_i had small effects on ligand binding. With 100 mM K⁺, 20 mM P_i modestly enhanced binding of ADP and hindered pyruvate binding but markedly enhanced the binding of pyruvate with ADP; the L_0.5 for pyruvate was specifically decreased ∼125-fold with 100 μM ADP. P_i 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.