Structure/function relationships in the pyruvate dehydrogenase complex form Azotobacter vinelandii

Abstract

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(pAPEl)] 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 (Elp) 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 boundlE2p monomer instead of one dimerE2p chain in trimeric wild-type E2p or E2p (pAPE1). The monomeric E2p species was catalytically inactive. Upon binding of excess Elp, some monomer formation of the E2p mutant took place. Elp however can prevent monomerization by E3. It is concluded that Elp 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 Elp 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 Elp or E3. Therefore, it seems likely that in vivo A. vinelundii 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 Elp or E3 seems unlikely. The results are discussed on the basis of the three-dimensional structure of the catalytic domain.Dihydrolipoyl transacetylase (E2p) is the core component Upon binding of the peripheral components pyruvate deof the pyruvate dehydrogenase complex (PDC) which cata-hydrogenase (Elp) and/or lipoamide dehydrogenase (E3), the lyzes the oxidative decarboxylation of pyruvate to acetyl-E2p from A. vinelandii behaves in a unique way as it disCoA [1,2]

Competitive Interaction of Component Enzymes with the Peripheral Subunit-Binding Domain of the Pyruvate Dehydrogenase Multienzyme Complex of Bacillus stearothermophilus: Kinetic Analysis Using Surface Plasmon Resonance Detection

“…In the octahedral branched chain 2-oxo acid dehydrogenase (BCDH) (Wynn et al, 1992) and icosahedral pyruvate dehydrogenase (PDH) (Perham, 1991;Mattevi et al, 1992;Lessard & Perham, 1995) complexes, the E1 component is bound to the peripheral subunit-binding domain of the E2 chain, whereas in the octahedral 2-oxoglutarate dehydrogenase (OGDH) and PDH complexes, a major part at least of the binding site for E1 resides in the acyltransferase (inner core) domain (Perham & Packman, 1989;Perham, 1991;Mattevi et al, 1992;Schulze et al, 1993). The peripheral subunit-binding domain is also responsible for binding the E3 component in all 2-oxo acid dehydrogenase complexes, irrespective of their symmetry (Perham, 1991;Mattevi et al, 1992;Hipps et al, 1994;Westphal et al, 1995), though in certain instances, this domain may be transposed from the E2 chain to protein X, an additional subunit found in low copy number in the E2 core of eukaryotic PDH complexes (Patel & Roche, 1990;Reed & Hackert, 1990 Maeng et al, 1996), or to the N terminus of the E1 component of mammalian OGDH complexes (Rice et al, 1992).…”

Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro

“…In contrast, the purified complex appears to contain 12 E1 dimers and 6 E3 dimers, perhaps as a result of steric hindrance between E1 and E3 [54]. However, the situation here is somewhat different in that the E1 component is not in direct competition with E3 for the peripheral-subunit binding domain of E2, but is bound at least in part by the acetyltransferase domain [2,9]. Steric factors may also play some part in the assembly of the icosahedral S. cerevisiae PDH complex, notably in the incorporation of E3BP, which is found in relatively few copies (estimated at about 12), despite the fact that each E2 chain in the icosahedral core has a potential binding site for it [13].…”

“…In the octahedral branched-chain 2-oxo acid-dehydrogenase (BCDH) [6] and icosahedral pyruvate-dehydrogenase (PDH) [7] complexes, the E1 component is bound to the peripheral subunitbinding domain of the E2 chain, whereas in the octahedral 2oxoglutarate-dehydrogenase (OGDH) and PDH complexes, a major part at least of the binding site for E1 resides in the acyltransferase (inner-core) domain [2,8,9]. The peripheral subunit-binding domain is also responsible for binding the E3 component in all 2-oxo acid-dehydrogenase complexes, irrespective of their symmetry [2,10,11], though in certain instances this domain may be located in protein X, an additional subunit found in low copy number in the E2 core of eukaryotic PDH complexes [1, 5, 12Ϫ14], or transposed to the N-terminus of the E1 component in mammalian OGDH complexes [15].…”