title: Investigation of the mechanism of active site coupling in the pyruvate dehydrogenase multienzyme complex of Escherichia coli by protein engineering.
abstract: Site-directed mutagenesis of the aceF gene of Escherichia coli was used to generate a nested set of deletions in the long (alanine + proline)-rich sequence that separates the lipoyl domain from the dihydrolipoamide dehydrogenase-binding domain in the "one-lipoyl domain" dihydrolipoamide acetyltransferase polypeptide chains of a pyruvate dehydrogenase multienzyme complex. The deletions reduced the number of residues in this sequence successively from 32 to 20, 13, 7 and just 1 residue. In all instances, pyruvate dehydrogenase complexes were still assembled in vivo around cores containing the deleted chains, and those with the two shortest deletions were essentially fully active. However, the two most severe deletions caused falls of 50% or more in specific catalytic activity. Similarly, although shortening the interdomain sequence to 20 residues left the system of active-site coupling unimpaired, cutting it to 13 residues or less caused substantial falls in the reductive acetylation of the lipoyl domains and corresponding losses of active-site coupling. The changes in specific catalytic activity and active-site coupling that accompanied the shortening of the (alanine + proline)-rich segment were reflected in the poorer growth rates of the relevant strains of E. coli on stringent substrates. All these results are consistent with this (alanine + proline)-rich sequence acting as a linker region that facilitates the movements of the lipoyl domains required for full catalytic activity and active-site coupling in the complex. The other two such sequences that separate the additional lipoyl domains in the N-terminal half of the wild-type "three-lipoyl domain" dihydrolipoamide acetyltransferase chain are presumed to function similarly. This role is consistent with the conformational flexibility assigned to these segments from previous studies based on 1H nuclear magnetic resonance spectroscopy and protein engineering.
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D009154:Mutation; D015202:Protein Engineering; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*
chemical_list: D011768:Pyruvate Dehydrogenase Complex