Purification and comparative study of the kinases specific for branched chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase.
Rat heart branched chain alpha-ketoacid dehydrogenase kinase (BCKDH kinase) and pyruvate dehydrogenase kinase (PDH kinase) were purified from their respective complexes to apparent homogeneity. BCKDH kinase consisted of one subunit with molecular weight 44,000-45,000 Da, whereas PDH kinase consisted of two subunits with molecular weight 48,000 Da (alpha) and 45,000 Da (beta) as previously shown for the bovine kidney enzyme (Stepp et al., 1983, J. Biol. Chem. 258, 9454-9458). Proteolysis maps of BCKDH kinase and the two subunits of PDH kinase were different, suggesting that all subunits are different entities. The alpha subunit of the rat heart PDH kinase could be cleaved selectively by chymotrypsin with concomitant loss of kinase activity, as previously shown for the bovine kidney enzyme, suggesting that the catalytic activity of PDH kinase resides in the alpha subunit. The beta subunit appeared to be a different entity unique to the PDH kinase. Both kinases exhibited marked substrate specificity toward their respective complexes and would not inactivate heterologous complexes. The kinases possessed slightly different substrate specificity toward histones. BCKDH kinase preferentially phosphorylated histones in the order f1 greater than f2B much greater than f2A much greater than f3. The relative order for PDH kinase was the same, but f2A and f3 were considerably better substrates than they were for BCKDH kinase. These observations suggest that the kinases have different requirements for the structure of the protein at their phosphorylation sites.
Popov
K M
KM
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Shimomura
Y
Y
Harris
R A
RA
D000818:Animals; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D006657:Histones; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D051381:Rats; D013379:Substrate Specificity
D006657:Histones; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C035627:(3-methyl-2-oxobutanoate dehydrogenase (lipoamide)) kinase