Sites of limited proteolysis in the pyruvate decarboxylase component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and their role in catalysis

Abstract

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...

Structure, mechanism and catalytic duality of thiamine-dependent enzymes

“…Our crystal structure of PDH-E1 from another thermophilic species, Bacillus stearothermophilus reveals a similar structural asymmetry of active site loops [26,39]. This asymmetry may account for earlier observations from solution studies, which show that one set of loops is preferentially susceptible to protease [40]. The structures of several other members of the ThDP enzyme family, available in the protein databank, also reveal conformational non-equivalences, hitherto unreported (summarized in Table 1).…”

“…Recently, we turned our attention to the B. stearothermophilus PDH-E1 in our attempts to explain the structural non-equivalence found in ThDP-dependent enzymes. Although this PDH-E1 contains two active sites, one is ordered while the other is not both in the crystal structure [26] and in solution [40]. Our attention was drawn to the middle of the protein, which contains a long cavity filled with at least fourteen hydrogen-bonded water molecules ( Fig.…”

Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy

“…The possibility that the movement of the lipoyl domain is not completely free but instead may occur via constrained trajectories has been suggested previously (41,42). Inspection of the outer surface of the icosahedral inner core of E2 reveals it to be predominantly positively charged, with a ring of positive charges near the entrance to each active site.…”

A Molecular Switch and Proton Wire Synchronize the Active Sites in Thiamine Enzymes

“…2A). Moreover, when B. stearothermophilus E1 is subjected to limited proteolysis, it is the outer of these loops ("275 to 293) that is the principal site of cleavage, but only half the E1 " chains appear to be susceptible, which implies a conformational asymmetry (14), as now observed in the crystal structure. In many other enzymes, active-site communication can be achieved through allosteric changes and subunit rearrangements (25); however, we did not find any significant structural differences between the subunits of B. stearothermophilus E1, other than the active-site loops.…”

“…The ThDP-dependent E1 component of the PDH multienzyme complex from Bacillus stearothermophilus has been intensively studied in this context (10,(12)(13)(14)(15). We have now solved the crystal structure of the subcomplex formed between the heterotetrameric (" 2 $ 2 ) B. stearothermophilus E1 and the peripheral subunit-binding domain (PSBD) from the lipoate acetyltransferase (E2) chain of the PDH complex (16).…”