Lipoylation of acyltransferase components of alpha-ketoacid dehydrogenase complexes.
Lipoic acid is a prosthetic group of the acyltransferase components of the pyruvate, alpha-ketoglutarate, and branched chain alpha-ketoacid dehydrogenase complexes, protein X of the eukaryotic pyruvate dehydrogenase complex, and H-protein of the glycine cleavage system. We have purified lipoyl-AMP:Nepsilon-lysine lipoyltransferase I and II from bovine liver mitochondria employing apoH-protein as an acceptor of lipoic acid (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1994) J. Biol. Chem. 269, 16605-16609). In this study, we demonstrated the lipoylation of the lipoyl domains of the mammalian pyruvate (LE2p), alpha-ketoglutarate (LE2k), and branched chain alpha-keto acid (LE2b) dehydrogenase complexes using the purified lipoyltransferase I and II. Lipoyltransferase I and II lipoylated LE2p and LE2k as efficiently as H-protein, but the lipoylation rate of LE2b was extremely low. Comparison of amino acid sequences surrounding the lipoylation site of these proteins shows that the conserved glutamic acid residue situated 3 residues to the N-terminal side of the lipoylation site is replaced by glutamine (Gln-41) in LE2b. When Gln-41 of LE2b was changed to Glu, the rate of lipoylation increased about 100-fold and became comparable to that of LE2p and LE2k. The replacement of the glutamic acid residue of LE2p (Glu-169) and LE2k (Glu-40) by glutamine resulted in decrease in the lipoylation rate more than 100-fold. These results suggest that the glutamic acid residue plays an important role in the lipoylation reaction possibly functioning as a recognition signal. Gly-27 and Gly-54 of LE2k are also well conserved among the lipoyl domains of the alpha-ketoacid dehydrogenase complexes and H-protein. The mutagenesis experiments of these residues indicated that the glycine residue situated 11 residues to the C-terminal side of the lipoylation site (Gly-54 of LE2k) is important for the folding of lipoyl domain, and that existence of a small residue such as Gly or Cys at the position is essential for the lipoylation of these proteins.
Fujiwara
K
K
Institute for Enzyme Research, the University of Tokushima, Tokushima 770, Japan.
Okamura-Ikeda
K
K
Motokawa
Y
Y
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism*
D018076:DNA, Complementary; D009097:Multienzyme Complexes; C067769:lipoyllysine; D018698:Glutamic Acid; D008063:Thioctic Acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000217:Acyltransferases; D008239:Lysine; D005998:Glycine