Repeating functional domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Abstract

Each polypeptide chain in the lipoate acetyltransferase (E2) core of the pyruvate dehydrogenase complex from Escherichia coli contains three repeating sequences in the N‐terminal half of the molecule. The repeats are highly homologous in primary structure and each includes a lysine residue that is a potential site for lipoylation. We have shown that all three sites are lipoylated, at least in part, and that the three lipoylated segments of the E2 chain can be isolated as distinct functional domains after limited proteolysis. Each domain becomes partly acetylated in the intact complex in the presence of substrate. In the primary structure, the domains are separated by regions of polypeptide chain oddly rich in alanine and proline residues. These regions are probably the conformationally mobile segments observed in the 1H‐n.m.r. spectrum of the complex and which are removed by tryptic cleavage at Lys‐316. The C‐terminal half of the molecule contains the acetyltransferase active site and the binding sites for E1, E3 and other E2 subunits. The pyruvate dehydrogenase complex of E. coli, which has a heterogeneous quaternary structure, is thus far unique among the 2‐oxo acid dehydrogenase complexes in possessing more than one lipoyl domain per E2 chain, but this may be a general feature of the enzyme from Gram‐negative organisms.

Apparent mitochondrial asymmetry in Xenopus eggs

“…The Xenopus PDC-E2 cDNA sequence contains all domains characteristic of dihydrolipoamide acetyltransferase (Bleile et al, 1981;Packman et al, 1984;De Marcucci et al, 1988;Thekkumkara et al, 1988;Russell and Guest, 1991;Matuda et al, 1992), including a mitochondrial targeting signal, two lipoyl-binding domains, a conserved E1/E3 subunit binding domain, and a CoA binding site within the conserved C-terminal catalytic domain (Fig. 5A).…”

Antibodies against an inter‐domain segment of polypeptide chain inhibit active‐site coupling in the pyruvate dehydrogenase multienzyme complex

“…PDH complexes were prepared from wild-type E. coli KI2 [16] and from a suitable deletion strain of E. coli transformed with plasmids pGS110 [14], pGSl56 [17] or pGS186-188 [15]. PDH complex activity, Elp and E3 partial activities and the acetyltransferase activity of the E2p component were measured as described [3,18].…”

“…The N-terminal half of the E2p chain contains a succession of three highly homologous lipoyl domains each of about 80 amino acids [2,3]; these are linked to each other and then to a smaller (approx. 50-residue) domain responsible, at least in part, for binding the E3 subunits [4,5].…”

“…50-residue) domain responsible, at least in part, for binding the E3 subunits [4,5]. The inter-domain segments (PEP1-3) of the polypeptide chain are long (20-30-residue) sequences rich in alanine, pro-line and charged amino acids [2,3]. The E3-binding domain is in turn linked by a shorter and less conspicuously (alanine+proline)-rich segment to a large (approx.…”

The functions of structure and dynamics in proteins, peptides and metal ion complexes and their relationships to biological recognition and the handling of information

“…Here I wish to stress the role of proline in the control of dynamics of activities. The first example is in stiffening single chain peptides which are part of hinged rods (1,12,26). The second is in the flipping between…”