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

C. W C Hu, T. A. Griffin, K. S. Lau, R. P. Cox, D. T. Chuang

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9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)343-349
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number1
StatePublished - Jul 9 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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