The pyruvate dehydrogenase multi-enzyme complex of Escherichia coli : genetic reconstruction and functional analysis of the lipoyl domains

Abstract

The dihydrolipoamide acetyltransferase (E2p) component of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous lipoyl domains ( ca . 100 residues) that are tandemly repeated to form the N-terminal half of the polypeptide chain. These lipoyl domains are linked to a much larger ( ca . 300 residues) subunit-binding domain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. Selective in vitro deletions in the E2p gene ( aceF )have allowed the creation of truncated E2p chains in which one or more of the lipoyl domains has been excised. Site-directed mutagenesis has been used to change individual residues. The effects of these deletions and mutations on the assembly, catalytic activity and active-site coupling in the complex are assessed.