The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was reconstituted in vitro from recombinant proteins derived from genes over-expressed in Escherichia coli. Titrations of the icosahedral (60-mer) dihydrolipoyl acetyltransferase (E2) core component with the pyruvate decarboxylase (E1, a 2 b 2 ) and dihydrolipoyl dehydrogenase (E3, a 2 ) peripheral components indicated a variable composition defined predominantly by tight and mutually exclusive binding of E1 and E3 with the peripheral subunit-binding domain of each E2 chain. However, both analysis of the polypeptide chain ratios in complexes generated from various mixtures of E1 and E3, and displacement of E1 or E3 from E1±E2 or E3±E2 subcomplexes by E3 or E1, respectively, showed that the multienzyme complex does not behave as a simple competitive binding system. This implies the existence of secondary interactions between the E1 and E3 subunits and E2 that only become apparent on assembly. Exact geometrical distribution of E1 and E3 is unlikely and the results are best explained by preferential arrangements of E1 and E3 on the surface of the E2 core, superimposed on their mutually exclusive binding to the peripheral subunit-binding domain of the E2 chain. Correlation of the subunit composition with the overall catalytic activity of the enzyme complex confirmed the lack of any requirement for precise stoichiometry or strict geometric arrangement of the three catalytic sites and emphasized the crucial importance of the flexibility associated with the lipoyl domains and intramolecular acetyl group transfer in the mechanism of active-site coupling.Keywords: multienzyme complex; pyruvate dehydrogenase complex; dihydrolipoyl acetyltransferase; selfassembly; competitive binding.The pyruvate dehydrogenase (PDH) multienzyme complex is a member of the family of 2-oxo acid dehydrogenase multienzyme complexes, all of which catalyse the oxidative decarboxylation of a 2-oxo acid and the reductive acylation of CoA, with concomitant reduction of NAD 1 and the release of CO 2 (reviewed in [1±3]). The complexes comprise multiple copies of three enzymes: a specific 2-oxo acid decarboxylase (E1); a specific dihydrolipoyl acyltransferase (E2); and dihydrolipoyl dehydrogenase (E3). In the PDH complex, E1 is pyruvate decarboxylase (EC 1.2.4.1), E2 is dihydrolipoyl acetyltransferase (EC 2.3.1.12) and E3 is dihydrolipoyl dehydrogenase (EC 1.8.1.4), which serves to reoxidize the dihydrolipoyl intermediate attached to E2 and exhibits no specificity towards the 2-oxo acid substrate.E2 forms the core of the multienzyme complex, to which E1 and E3 bind tightly but noncovalently. E1 may be dimeric (a 2 ) or heterotetrameric (a 2 b 2 ), depending on the source, and E3 is a dimer (a 2 ). The E2 polypeptide chain is composed of three types of domain, separated by long, flexible linker regions: one to three lipoyl domains (9 kDa) at the N-terminus (the number depending on the source of the complex); a small peripheral (E1 and/or E3) subunit-binding domain (PSBD, 4 k...
Limited proteolysis of the pyruvate decarboxylase (E1, alpha2beta2) component of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1alpha subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. & Perham, R.N. (2000) Eur. J. Biochem. 267, 7158-7169). This site appears to be conserved in the alpha-subunit of heterotetrameric E1s and multiple sequence alignments suggest that there are additional conserved amino-acid residues in this region, part of a common pattern with the consensus sequence -YR-H-D-YR-DE-. This region lies about 50 amino acids on the C-terminal side of a 30-residue motif previously recognized as involved in binding thiamin diphosphate (ThDP) in all ThDP-dependent enzymes. The role of individual residues in this set of conserved amino acids in the E1alpha chain was investigated by means of site-directed mutagenesis. We propose that particular residues are involved in: (a) binding the 2-oxo acid substrate, (b) decarboxylation of the 2-oxo acid and reductive acetylation of the tethered lipoyl domain in the PDH complex, (c) an "open-close" mechanism of the active site, and (d) phosphorylation by the E1-specific kinase (in eukaryotic PDH and branched chain 2-oxo acid dehydrogenase complexes).
Heteronuclear NMR spectroscopy and other experiments indicate that the true substrate of the E1 component of 2-oxo acid dehydrogenase complexes is not lipoic acid but the lipoyl domain of the E2 component. E1 can recognize the lipoyl-lysine residue as such, but reductive acylation ensues only if the domain to which the lipoyl group is attached is additionally recognized by virtue of a mosaic of contacts distributed chiefly over the half of the domain that contains the lipoyl-lysine residue. The lipoyl-lysine residue may not be freely swinging, as supposed hitherto, but may adopt a preferred orientation pointing towards a nearby loop on the surface of the lipoyl domain. This in turn may facilitate the insertion of the lipoyl group into the active site of E1, where reductive acylation is to occur. The results throw new light on the concept of substrate channelling and active-site coupling in these giant multifunctional catalytic machines.
The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (a 2 b 2 ) of E1a and E1b polypeptide chains. The domain structure of the E1a and E1b chains, and the protein±protein interactions involved in assembly, have been studied by means of limited proteolysis. It appears that there may be two conformers of E1a in the E1 heterotetramer, one being more susceptible to proteolysis than the other. A highly conserved region in E1a, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (a 2 b 2 ). As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchanged. The parameters of the interaction between cleaved E1 and the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild-type E1. However, a pyruvate dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1. This implies that active site coupling between the E1 and E2 components has been impaired. This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.Keywords: pyruvate decarboxylase; pyruvate dehydrogenase; multienzyme complex; limited proteolysis; enzyme catalysis.2-Oxo acid dehydrogenase multienzyme complexes consist of multiple copies of three component enzymes that bring about the oxidative decarboxylation of 2-oxo acids and the transfer of the resulting acyl group to coenzyme A. In the pyruvate dehydrogenase (PDH) complexes of Gram-negative bacteria, such as Escherichia coli, these enzymes are normally found as three different types of polypeptide chain, corresponding to the pyruvate decarboxylase (E1p; EC 1.2.4.1), dihydrolipoyl acetyltransferase (E2p; EC 2.3.1.12) and dihydrolipoyl dehydrogenase (E3; EC 1.8.1.4) components, and the E2p core consists of 24 copies of the E2p chain arranged with octahedral symmetry. On the other hand, the PDH complexes of Gram-positive bacteria, such as Bacillus stearothermophilus, and of mammals contain four different types of polypeptide chain: E1a and E1b (which form E1p, a 2 b 2 ), E2p and E3; and the E2p core consists of 60 copies of the E2p chain arranged with icosahedral symmetry (reviewed [1±3]).The B. stearothermophilus E2p chain consists of three domains separated by flexible linker regions: an N-terminal lipoyl domain that hosts the specific lysine residue (Lys42) to which the lipoyl group is attached; a peripheral subunit-binding domain (PSBD) [4], responsible for the tight but noncovalent binding of E1p and E3; and a C-terminal acetyltransferase domain responsible for assembly of the 60-mer icosahedral inner core [5] and catalytic transfer of the acetyl group to coenzyme A [1,6]. The E3 component, a ho...
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