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

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 approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 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 K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 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.

Yeast Pyruvate Dehydrogenase Complex Is Regulated by a Concerted Activity of Two Kinases and Two Phosphatases

“…46). These regulatory enzymes themselves are regulated by a variety of conditions, including the intramitochondrial concentrations of cofactors and substrates or products of PDC reaction (1,3,7,11,(47)(48)(49), indicating a complex multilayer regulatory network. Failure of the human PDC (50) or of one of its regulatory enzymes (51,52) results in serious disorders.…”

Ligand-induced Effects on Pyruvate Dehydrogenase Kinase Isoform 2

“…We also demonstrate that the binding of pyruvate and ADP or ATP alters the oligomeric state of PDHK2. These studies provide new insights into the mechanistic bases for the synergistic action of ADP plus pyruvate in down-regulating PDHK2 activity (27).…”

“…Attenuation of PDHK2 activity is primarily by ADP (an indicator of low energy state) and pyruvate (an indicator of substrate sufficiency). These effectors act synergistically to inhibit PDHK activity (26,27). Recently, we presented evidence that PDHK2 catalysis is limited by ADP dissociation and that having ADP bound favors the development of inhibition by pyruvate binding to the PDHK2⅐ADP intermediate (27).…”

“…The capacity to characterize the E2-enhanced physiological PDHK2 reaction by kinetic studies is limited by the nonequilibrium nature of the catalytic steps. The first substrate (ATP) dissociates slower than k cat (27). The second substrate, E1, is delivered within the confines of complex, in part, by nondissociative mechanisms (2,7,8,15), and is converted through a two-step series to becoming alternative substrate 1 and then alternative substrate 2.…”

“…These effectors act synergistically to inhibit PDHK activity (26,27). Recently, we presented evidence that PDHK2 catalysis is limited by ADP dissociation and that having ADP bound favors the development of inhibition by pyruvate binding to the PDHK2⅐ADP intermediate (27). Furthermore, stimulation by reductive acetylation of lipoyl groups was linked to an increase in speed in the dissociation of ADP (21).…”