Characterization and conservation of the inner E2 core domain structure of branched-chain α-keto acid dehydrogenase complex from bovine liver. Construction of a cDNA encoding the entire transacylase (E(2b)) precursor

T. A. Griffin, K. S. Lau, D. T. Chuang

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Abstract

A cDNA clone encoding the entire transacylase (E(2b)) precursor of the bovine branched-chain α-keto acid dehydrogenase complex has been constructed from two overlapping incomplete cDNA clones which were isolated from a λZAP library prepared from bovine liver poly(A)+ RNA. Nucleotide sequencing indicates that this bovine E(2b) cDNA insert (bE2-11) is 2701 base pairs in length with an open reading frame of 1446 base pairs. The bE2-11 cDNA insert encodes a leader peptide of 61 residues and a mature E(2b) polypeptide of 421 amino acid residues with a calculated monomeric molecular mass of 46,518 daltons. The molecular mass of the native E(2b) component isolated from bovine liver is 1,110,000 daltons as determined by sedimentation equilibrium. This value establishes the 24-subunit octahedral model for the quaternary structure of bovine E(2b). The amino-terminal sequences of two tryptic fragments (A and B) of the E(2b) protein have been determined. Fragment A comprises residues 175 to 421 of the E(2b) protein and is the inner E2 core domain which contains the transacylase active site. Fragment B, produced by further tryptic cleavage of fragment A, comprises residues 205 to 421, but does not have transacylase activity. Both fragments A and B confer the highly assembled 24-mer structure. The primary structure of the inner E2 core domain of bovine E(2b) (fragment A) is very similar to those of three other E2 proteins (human E(2p), Escherichia coli E(2p), and E. coli E(2k)). These similarities suggest that these E2 proteins are structurally and evolutionarily related.

Original languageEnglish (US)
Pages (from-to)14008-14014
Number of pages7
JournalJournal of Biological Chemistry
Volume263
Issue number28
StatePublished - Jan 1 1988

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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