Subunit structure of the dihydrolipoyl transacylase component of branched-chain α-keto acid dehydrogenase complex from bovine liver: mapping of the lipoyl-bearing domain by limited proteolysis

To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain α-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C]α-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (M/sub r/ = 52,000) into five radiolabeled lipoyl-containing fragments, L1-L5. In addition, a lipoate-free inner E2 core consisting of fragment A and fragment B was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserium against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum-strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain