Site‐directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Abstract

(Receivcd May 13, 1991) -EJB 91 0615Site-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 (Elp) 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 '4. vinelandii wild-type E2p dissociates into tetramers. Four E l p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E l p dimers.Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E l p and E3 binding. A small deletion ( A P361-R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E l p binding strongly, although with excess Elp 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 Elp/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 111 from Eschevichia 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 E l p 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. E l p 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 E l p with the E l p 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 E l p 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.The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA [I]. It

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli

“…These experiments confirm our previous conclusion [14], based on site-directed mutagenesis, that the E l p sites consist of amino acids derived from both the binding and the catalytic domain. The point mutations Lys367 -P Gln (binding domain) and Arg416 --f Asp (catalytic domain) dramatically decreased the affinity for Elp, but not for E3 at catalytically significant ratios.…”

“…Competition exists between E3, added in excess with respect to its own sites, and E l p for binding to the E l p sites. This is not merely a steric effect due to occupation of the binding domain, because the mutations at the N-terminal part of the catalytic domain also affected E3 binding, when added in excess [14]. On the other hand, it could be shown that the peripheral components do not bind to the isolated catalytic domain of A .…”

“…In this case this may be due to a spacing problem, caused by the removal of the apa-4 sequence. However, other mutants in which this sequence was deleted (pAPE1 and pAPE2 [14]) had normal binding properties. This indicates that E3, when binding to E l p sites also interacts with the catalytic domain.…”

“…vinelandii and E. coli with the chimeric E2p proteins. Binding of E l p and/or E3 to the E2p core was monitored on a Superose-6 gel-filtration column (FPLC) in 50 mM potassium phosphate pH 7.0 containing 0.5 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride (PhMeS02F) as described in [14]. Alternatively, binding was studied by sedimentation analysis of the chimeric E2p proteins and of E2p/E3 or E2p/Elp subcomplexes.…”

“…In E2p from A . vinelandii this region is concerned in the binding of the peripheral components E3 and E l p [13,14]. By site-directed mutagenesis it was shown that the N-terminal part of the catalytic domain is also part of the Elp binding site [14].…”