Studies on the regulation of the mitochondrial alpha-ketoacid dehydrogenase complexes and their kinases.
Five mitochondrial protein kinases, all members of a new family of protein kinases, have now been identified, cloned, expressed as recombinant proteins, and partially characterized with respect to catalytic and regulatory properties. Four members of this unique family of eukaryotic protein kinases correspond to pyruvate dehydrogenase kinase isozymes which regulate the activity of the pyruvate dehydrogenase complex, an important regulatory enzyme at the interface between glycolysis and the citric acid cycle. The fifth member of this family corresponds to the branched-chain alpha-ketoacid dehydrogenase kinase, an enzyme responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex, the most important regulatory enzyme in the pathway for the disposal of branched-chain amino acids. At least three long-term control mechanisms have evolved to conserve branched chain amino acids for protein synthesis during periods of dietary protein insufficiency. Increased expression of the branched-chain alpha-ketoacid dehydrogenase kinase is perhaps the most important because this leads to phosphorylation and nearly complete inactivation of the liver branched-chain alpha-ketoacid dehydrogenase complex. Decreased amounts of the liver branched-chain alpha-ketoacid dehydrogenase complex secondary to a decrease in liver mitochondria also decrease the liver's capacity for branched-chain keto acid oxidation. Finally, the number of E1 subunits of the branched-chain alpha-ketoacid dehydrogenase complex is reduced to less than a full complement of 12 heterotetramers per complex in the liver of protein-starved rats. Since the E1 component is rate-limiting for activity and also the component of the complex inhibited by phosphorylation, this decrease in number further limits overall enzyme activity and makes the complex more sensitive to regulation by phosphorylation in this nutritional state. The branched-chain alpha-ketoacid dehydrogenase kinase phosphorylates serine 293 of the E1 alpha subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Site-directed mutagenesis of amino acid residues surrounding serine 293 reveals that arginine 288, histidine 292 and aspartate 296 are critical to dehydrogenase activity, that histidine 292 is critical to binding the coenzyme thiamine pyrophosphate, and that serine 293 exists at or in close proximity to the active site of the dehydrogenase. Alanine scanning mutagenesis of residues in the immediate vicinity of the phosphorylation site (serine 293) indicates that only arginine 288 is required for recognition of serine 293 as a phosphorylation site by the branched-chain alpha-ketoacid dehydrogenase kinase. Phosphorylation appears to inhibit dehydrogenase activity by introducing a negative charge directly into the active site pocket of the E1 dehydrogenase component of the branched-chain alpha-ketoacid dehydrogenase complex. A model based on the X-ray crystal structure of transketolase is being used to predict residues involved in thiamine pyrophosphate binding and to help visualize how phosphorylation within the channel leading to the reactive carbon of thiamine pyrophosphate inhibits catalytic activity. The isoenzymes of pyruvate dehydrogenase kinase differ greatly in terms of their specific activities, kinetic parameters and regulatory properties. Chemically-induced diabetes in the rat induces significant changes in the pyruvate dehydrogenase kinase isoenzyme 2 in liver. Preliminary findings suggest hormonal control of the activity state of the pyruvate dehydrogenase complex may involves tissue specific induced changes in expression of the pyruvate dehydrogenase kinase isoenzymes.
Harris
R A
RA
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122, USA.
Hawes
J W
JW
Popov
K M
KM
Zhao
Y
Y
Shimomura
Y
Y
Sato
J
J
Jaskiewicz
J
J
Hurley
T D
TD
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004032:Diet; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013835:Thiamine Pyrophosphate / Q000378:metabolism / Q000494:pharmacology
D009097:Multienzyme Complexes; C000631436:Pdk2 protein, rat; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C035627:(3-methyl-2-oxobutanoate dehydrogenase (lipoamide)) kinase; D013835:Thiamine Pyrophosphate