Site-directed mutagenesis within the catalytic domain of pyruvate dehydrogenase kinase (PDK)

Academic Article


  • Five conserved subdomains resembling the subdomains of "sensor" kinases engaged in the regulation of the adaptive response in bacteria have been identified within the amino acid sequence of PDK through a comparison of the primary structures of mitochondrial protein kinases. Subdomain 1 defined by invariant H115 (numbered according to the sequence of rat isoenzyme 2 of PDK); subdomain 2 defined by N247; subdomain 3 corresponding to the consensus sequence D282×G284×G286; subdomain 4 - Y298; and subdomain 5 defined by consensus sequence G317×G319×G321. In order to evaluate the functional roles of these subdomains in the catalysis of the kinase reaction, the invariant N247, D282, G284, G286 and G319 residues of rat PDK2 were substituted to alanine and invariant H115 residue to serine by site-directed mutagenesis. The respective mutant cDNAs were expressed in Escherichia coll and corresponding mutant proteins purified by metal chelate Chromatograph y and characterized. Substitution of N274, D282, and G286 produced proteins without measurable enzymatic activities, whereas the G284 and G319 mutants had the specific activities comparable to that of wild type enzyme. However, the Km for ATP of G319 mutant was increased up to 400 times. The G284 mutant, in contrast, had the same affinity for ATP as wild type enzyme, but did not demonstrate inhibition by ADP. Substitution of H115, the residue homologous to the autophosphorylation site of bacterial "sensor" kinases, to serine produced a fully active enzyme. Therefore, the results suggest that PDK as well as other mitochondrial protein kinases are similar with their bacterial counterparts in terms of general organization of ATP-binding domains located within carboxyl terminus of the kinase molecule, but utilize a different mechanism of phosphotransfer.
  • Published In

  • The FASEB Journal  Journal
  • Author List

  • Bowker-Kinlev M; Popov KM
  • Volume

  • 10
  • Issue

  • 6