PURIFICATION AND CHARACTERIZATION OF LIPOYL-AMP-N-EPSILON-LYSINE LIPOYLTRANSFERASE FROM BOVINE LIVER-MITOCHONDRIA

Lipoyl-AMP:N-epsilon-lysine lipoyltransferase (lipoyltransferase) cata lyzes the transfer of the lipoyl group from lipoyl AMP to a lysine res idue of the specific enzyme proteins. We have shown previously that th e lipoyltransferase activities locate in mitochondria using apoH-prote in of the glycine cleavage system as an acceptor of the lipoyl group ( Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1990) J. Biol. Chem . 265, 17463-17467). Here we describe the purification and the charact erization of two isoforms of lipoyltransferase termed lipoyltransferas e I and lipoyltransferase II from bovine liver mitochondria. Lipoyltra nsferase II was purified to apparent homogeneity, whereas the final pr oduct of lipoyltransferase I still contained a minor contaminant. Alth ough the two forms could be resolved on a hydroxylapatite column chrom atography, they were indistinguishable, as judged by: (a) behavior dur ing purification on ion exchange, hydrophobic, or affinity columns; (b ) molecular mass determined by sodium dodecyl sulfate-polyacrylamide g el electrophoresis and gel exclusion chromatography (40 kDa); and (c) catalytic properties (substrate specificity, kinetic constants, and op timal pH), Both lipoyltransferase I and II could not use lipoic acid p lus MgATP as a substrate in place of lipoyl-AMP. Surprisingly, the lip oyltransferases transferred not only the lipoyl group but also the acy l groups from hexanoyl-, octanoyl-, and decanoyl-AMP to apoH-protein t o a similar extent.