Abstract
Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/L0.5 estimates: approximately 53%/3 microM for ATP; approximately 49%/15 microM for ADP; and approximately 71%/approximately 590 microM for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and > or = 80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 microM pyruvate, > or = 150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2(red)) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2(red) was modestly hindered by 200 microM level of ATP or ADP or 5.0 mM pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 microM levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2(red) may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.
Highlights
Because this results in animals in the irrecoverable reduction of body carbohydrate reserves, pyruvate dehydrogenase complex (PDC) activity is tightly regulated by dedicated regulatory enzymes [1, 2]
We demonstrate that the binding of pyruvate and ADP or ATP alters the oligomeric state of PDHK2
Pyruvate plus ADP markedly decreased binding of PDHK2 to glutathione S-transferase (GST)-L2 dimer and caused the PDHK2 dimer to associate to form a loose tetramer
Summary
Materials—Recombinant human PDHK2 [12], E2 [3], GST-L2 [38], and E3 [39] were prepared as previously described. Quenching studies that included pyruvate or DCA, whether added alone or in combination with ATP or ADP, yielded nonhyperbolic profiles for the decrease in fluorescence with increasing concentration of the varied ligand. In those cases, Qmax values were estimated from a double reciprocal plot using the higher concentrations and the data plotted in Hill plots (log(Y/1 Ϫ Y) versus log(Lf)) or plots of log(Y) versus log(Lf) with the latter made and analyzed by the Sigma Plot program. Sedimentation equilibrium studies were conducted on PDHK2 in the presence of 100 M ADP and 100 M pyruvate with PDHK2 added at concentrations of 1.70, 2.43, and 3.06 M PDHK2 dimer in buffer A. Sedimentation equilibrium data were globally fit to monomer-dimer and monomer-trimer models using Beckman software (version 4.0) that was provided with the Optima XL-I ultracentrifuge
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