The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component

Abstract

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.

Three-dimensional Structure of the Lipoyl Domain from the Dihydrolipoyl Succinyltransferase Component of the 2-Oxoglutarate Dehydrogenase Multienzyme Complex ofEscherichia coli

“…In both instances, the protein was found as the apo-domain (unlipoylated), as noted previously for the lipoyl domain from the E2p chain of B. stearothermophilus (Hipps et al, 1994). This is thought to be due to the over-expression of the domain swamping the lipoylation machinery of the E. coli cell.…”

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

“…We have shown previously that one peripheral subunitbinding domain is capable of binding to one E1 tetramer (R 2 2 ) or one E3 dimer but not to both simultaneously (Hipps et al, 1994;Lessard & Perham, 1995). We have now obtained precise measurements of the kinetics of the interac- (8, 10, 12, 14, 17, 20, and 25 nM).…”

“…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).…”

Recognition of a Surface Loop of the Lipoyl Domain Underlies Substrate Channelling in the Pyruvate Dehydrogenase Multienzyme Complex

“…The cells were induced for three hours with IPTG (final concentration 1 mM) and harvested by centrifugation at 5000 g and 4°C for five minutes. 15 N-labelled ThDD was purified from the cell-free extracts, essentially as described for the natural di-domain by Hipps et al (1994). A solution of ThDD (10 mg/ml) in 20 mM potassium phosphate buffer (pH 7.0) was digested with chymotrypsin (0.1% (w/v) final concentration), at 37°C for six hours.…”

“…The E1 component decarboxylates the 2-oxo acid and reductively acylates a lipoyl-lysine residue in E2, which functions as a swinging arm to transfer the substrate between the three active sites of the enzyme complex. The E2 polypeptide chain is made up of three types of domain arrayed in tandem and separated by flexible linker regions: at the N terminus are one to three lipoyl domains (Reed & Hackert, 1990;Perham, 1991); on the C-terminal side of the lipoyl domain lies a much smaller domain responsible for binding the E3 and (in icosahedral complexes) E1 subunits (Hipps et al, 1994;Lessard & Perham, 1995;Westphal et al, 1995); and the C-terminal region comprises the large (28 kDa) inner-core domain which contains the acyltransferase active site and aggregates to form the octahedral (24-mer, as in Escherichia coli PDH) or icosahedral (60-mer, as in Bacillus stearothermophilus PDH) inner core of the complex (Reed & Hackert, 1990;Perham, 1991).…”