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

Abstract
The dihydrolipoamide acetyltransferase (E2p) component of the pyruvate dehydrogenase complex ofEscherichia colicontains 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. Selectivein vitrodeletions 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.