Site-directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Binding of the peripheral components E1p and E3.
Site-directed mutagenesis was performed in the protease-sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex. Upon binding of peripheral components, the 24-subunit core of A. vinelandii wild-type E2p dissociates into tetramers. Four E1p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers. Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E1p and E3 binding. A small deletion (delta P361-R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached. For E2p with the point mutation R416D in the N-terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli. Deletion of the complete Ala + Pro-rich sequence between the protease-sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3. A further deletion of 20 N-terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E1p binding was lost and, in contrast to the wild-type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes. It is concluded that the binding site for E3 is located on the N-terminal part of the protease-sensitive region. In contrast, the binding site for E1p consists of two regions, one located on the protease-sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids.