An Essential Role of Glu-243 and His-239 in the Phosphotransfer Reaction Catalyzed by Pyruvate Dehydrogenase Kinase

Academic Article


  • This study was undertaken to examine the mechanistic significance of two highly conserved residues positioned in the active site of pyruvate dehydrogenase kinase, Glu-243 and His-239. We used site-directed mutagenesis to convert Glu-243 to Ala, Asp, or Gln and His-239 to Ala. The resulting mutant kinases demonstrated a greatly reduced capacity for phosphorylation of pyruvate dehydrogenase. The Glu-243 to Asp mutant had ∼2% residual activity, whereas the Glu-243 to Ala or Gln mutants exhibited less than 0.5 and 0.1% residual activity, respectively. Activity of the His-239 to Ala mutant was decreased by ∼90%. Active-site titration with [α-32PIATP revealed that neither Glu-243 nor His-239 mutations affected nucleotide binding. All mutant kinases showed similar or even somewhat greater affinity than the wild-type kinase toward the protein substrate, pyruvate dehydrogenase complex. Furthermore, neither of the mutations affected the inter-subunit interactions. Finally, pyruvate dehydrogenase kinase was found to possess a weak ATP hydrolytic activity, which required Glu-243 and His-239 similar to the kinase activity. Based on these observations, we propose a mechanism according to which the invariant glutamate residue (Glu-243) acts as a general base catalyst, which activates the hydroxyl group on a serine residue of the protein substrate for direct attack on the γ phosphate. The glutamate residue in turn might be further polarized through interaction with the neighboring histidine residue (His-239).
  • Published In

    Digital Object Identifier (doi)

    Author List

  • Tuganova A; Yoder MD; Popov KM
  • Start Page

  • 17994
  • End Page

  • 17999
  • Volume

  • 276
  • Issue

  • 21