Structure/function relationships in the pyruvate dehydrogenase complex from Azotobacter vinelandii. Role of the linker region between the binding and catalytic domain of the dihydrolipoyl transacetylase component.
The role of the hinge region between the binding domain and the catalytic domain in dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii was addressed by deletion mutagenesis. Mutated dihydrolipoyl transacetylase proteins were constructed with a deletion of 11 amino acids in the hinge region between the binding domain and the N-terminal part of the catalytic domain of E2p [E2p(pAPE1)] and with a further deletion of 9 amino acids into the N-terminal sequence protruding from the globular structure of the catalytic domain [E2p(pAPE2)] and found to take part in the intratrimer interaction. Both proteins behaved as wild-type E2p with respect to catalytic activity and quaternary structure. The interaction of the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) with the mutated E2p proteins was studied. E2p(pAPE1) assembles to a trimeric pyruvate dehydrogenase complex (PDC) with 15% decreased complex activity. No difference in affinity towards the peripheral components was detected. Upon binding of E3, E2p(pAPE2) dissociates into trimers and monomers. At saturation, two dimers of E3 were bound/E2p monomer instead of one dimer/E2p chain in trimeric wild-type E2p or E2p (pAPE1). The monomeric E2p species was catalytically inactive. Upon binding of excess E1p, some monomer formation of the E2p mutant took place. E1p however can prevent monomerization by E3. It is concluded that E1p is bound between two different E2p chains in the trimer. The substrates CoA and acetyl-CoA also prevent monomerization because they are bound by amino acid residues of two different E2p chains. In the presence of CoA no difference in affinity with respect to E1p and E3 binding was observed. CoA (and acetylCoA) also prevent dissociation of the 24-subunit core structure of wild-type E2p when added before addition of E1p or E3. Therefore, it seems likely that in vivo A. vinelandii PDC is based on a 24-subunit E2p core, like Escherichia coli PDC. A functional difference between complexes based on a trimer or a 24-subunit core has not been observed. A role of the hinge region as a spacer to allow binding of E1p or E3 seems unlikely. The results are discussed on the basis of the three-dimensional structure of the catalytic domain.
Schulze
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Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Westphal
A H
AH
Hanemaaijer
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de Kok
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A
D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D002850:Chromatography, Gel; D003065:Coenzyme A / Q000494:pharmacology; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D017353:Gene Deletion; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D003065:Coenzyme A