Pyruvate Dehydrogenase Kinase Isoform 2 Activity Limited and Further Inhibited by Slowing Down the Rate of Dissociation of ADP

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Abstract
Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K+ at ∼90 mM and Cl- at ∼60 mM decreased the Km of PDK2 for ATP and competitive Ki for ADP by ∼3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis ± E2, E2 increased the Km of PDK2 for ATP by nearly 8-fold (from 5 to 39 μM), increased kcat by ∼4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a Kd of 5 μM, which equals the Km and Kd of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had ∼3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2·[ADP or ATP]·pyruvate complexes form, and that PDK2·ATP·pyruvate·E1 reacts with PDK2·ADP·pyruvate accumulating.

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