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 phyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E₂ polypeptide chain [(M(r) = 52,000] into five radiolabeled lipoyl-containing fragments in the order of L₁ [M(r) = 28,000], L₂ [M(r) = 24,500], L₃ [M(r) = 21,000], L₄ [M(r) = 15,000] to L₅ [M(r) = 14,000] as determined by the autoradiogrpahy of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E₂ core consisting of fragment A [M(r) = 26,000] and fragment B [M(r) = 22,000] 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 antiserum 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. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E₂ core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L₁ to L₅), with the L₁ site in the hinge region, and the L₅ site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L₁ and L₄ tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E₂ chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L₅, the limit polypeptide carrying the single lipoyl residue.