Production of Acetyl-CoA (Activated Acetate):- The Pyruvate Dehydrogenase Complex Consists of Three Distinct Enzymes
The PDH complex contains three enzymes—pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3)—each present in multiple copies. The number of copies of each enzyme and therefore the size of the complex varies among species. The PDH complex isolated from mammals is about 50 nm in diameter—more than five times the size of an entire ribosome and big enough to be visualized with the electron microscope (Fig. 16–5a). In the bovine enzyme, 60 identical copies of E2 form a pentagonal dodecahedron (the core) with a diameter of about 25 nm (Fig. 16–5b). (The core of the Escherichia coli enzyme contains 24 copies of E2.) E2 is the point of connection for the prosthetic group lipoate, attached through an amide bond to the -amino group of a Lys residue (Fig. 16–4). E2 has three functionally distinct do mains (Fig. 16–5c): the amino-terminal lipoyl domain, containing the lipoyl-Lys residue(s); the central E1- and E3-binding domain; and the inner-core acyltransferase domain, which contains the acyltransferase active site. The yeast PDH complex has a single lipoyl domain with a lipoate attached, but the mammalian complex has two, and E. coli has three (Fig. 16–5c). The domains of E2 are separated by linkers, sequences of 20 to 30 amino acid residues, rich in Ala and Pro and interspersed with charged residues; these linkers tend to assume their ex tended forms, holding the three domains apart. The active site of E1 has bound TPP, and that of E3 has bound FAD. Also, part of the complex are two regulatory proteins, a protein kinase and a phosphoprotein phosphatase, discussed below. This basic E1- E2 -E3 structure has been conserved during evolution and used in a number of similar metabolic reactions, including the oxidation of -ketoglutarate in the citric acid cycle (described below) and the oxidation of -keto acids derived from the breakdown of the branched-chain amino acids valine, isoleucine, and leucine. Within a given species, E3 of PDH is identical to E3 of the other two enzyme complexes. The attachment of lipoate to the end of a Lys side chain in E2 produces a long, flexible arm that can move from the active site of E1 to the active sites of E2 and E3, a distance of perhaps 5 nm or more.
FIGURE 16–5 Structure of the pyruvate dehydrogenase complex (a) Cryoelectron micrograph of PDH complexes isolated from bovine kidney. In cryoelectron microscopy, biological samples are viewed at extremely low temperatures; this avoids potential artifacts introduced by the usual process of dehydrating, fixing, and staining. (b) Three-dimensional image of PDH complex, showing the subunit structure: E1, pyruvate dehydrogenase; E2, dihydrolipoyl transacetylase; and E3, dihydrolipoyl dehydrogenase. This image is reconstructed by analysis of a large number of images such as those in (a), combined with crystallographic studies of individual subunits. The core (green) consists of 60 molecules of E2, arranged in 20 trimers to form a pentagonal dodecahedron. The lipoyl domain of E2 (blue) reaches outward to touch the active sites of E1 molecules (yellow) arranged on the E2 core. A number of E3 subunits (red) are also bound to the core, where the swinging arm on E2 can reach their active sites. An asterisk marks the site where a lipoyl group is attached to the lipoyl domain of E2. To make the structure clearer, about half of the complex has been cut away from the front. This model was prepared by Z. H. Zhou et al. (2001); in another model, proposed by J. L. S. Milne et al. (2002), the E3 subunits are located more toward the periphery (see Further Reading). (c) E2 consists of three types of domains linked by short polypep tide linkers: a catalytic acyltransferase domain; a binding domain, in volved in the binding of E2 to E1 and E3; and one or more (depending on the species) lipoyl domains.
