Mammalian pyruvate dehydrogenase complex (PDC) is a multienzyme complex composed of three catalytic components, namely pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3); one structural protein called E3-binding protein (BP) and two specific families of regulatory enzymes; namely pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs). Three catalytic components of PDC catalyze the oxidative decarboxylation of pyruvic acid with formation of carbon dioxide, acetyl-CoA, NADH and H+
. E1 carries out the decarboxylation of pyruvate and reductive acetylation of the lipoyl moieties of E2. E2 then transfers the acetyl moiety to CoA forming acetyl-CoA. E3 catalyzes reoxidation of the reduced lipoyl moieties of E2 with the reduction of NAD+
to NADH [1
]. PDC plays a key role in the maintenance of glucose homeostasis, and its activity is tightly regulated through the phosphorylation/dephosphorylation catalyzed by PDKs and PDPs.
PDC is a highly organized multienzyme complex. E2 and BP (in higher eukaryotes and only E2 in other organisms) form the core of the complex and bind peripheral components, i.e. E1, E3, PDKs and PDPs in higher eukaryotes [2
]. In mammals 20–30 heterotetramers of E1 (α2
) are bound to the E2; 6–12 homodimers of E3 are bound to BP; 1–3 copies of PDK and 2–3 copies of PDP are bound to E2 and/or BP [4
]. The PDC core has icosahedral symmetry in eukaryotes and some Gram-positive bacteria and octahedral in Gram-negative bacteria [4
]. E2 and BP have similar structures composed of three structural domains connected by flexible hinge regions: (i) the lipoyl domains [two for human PDC-E2, named L1, the outer domain and L2, the inner domain and one for human (h) PDC-BP, named L3]; (ii) the subunit-binding domain interacting with E1 and/or E3 and (iii) the inner domain, forming the central core of PDC and carrying out the catalytic reaction of E2 () [3
]. In bacterial PDC both E1 and E3 are bound to the subunit-binding domains of E2. In higher eukaryotes E1 is bound to the subunit-binding domain of E2 and E3 to the subunit-binding domain of BP ().
Binding domains of E1 and E3 in different PDCs. L, L1, L2, L3 are lipoyl domains of bacterial E2 and human E2 and BP. Sb, S and S1 are the corresponding subunit-binding domains.
Human E1, an α2
heterotetramer, has two active sites. Based on the 3D structure, the two active sites of hE1 are proposed to interact with each other during catalysis by a ‘flip-flop mechanism’ [6
]. E1s of icosahedral PDCs having similar structures to the hE1 heterotetrameric structure (α2
) bind to their cognate E2s through the C-terminal of their β subunits [7
]. The structure of the subcomplex of Bacillus stearothermophilus (bs)
E1 with the E1/E3-binding domain of bsE2 was determined by Frank et al. [7
]. Recently we reported several residues participating in binding of hE1 to the E1-binding domain of hE2 [8
], and these findings are summarized here for comparison. E1s of octahedral PDCs which are homodimers bind to E2 through their N-terminal regions as was found for E. coli
and Azotobacter vinelandii
E3, the product of a single Dld
gene in eukaryotes, serves as a component of three α-keto acid dehydrogenase complexes [PDC, branched-chained α-ketoacid dehydrogenase (BCKDH) complex and α-ketoglutarate dehydrogenase complex] and also as L protein in the glycine cleavage system [2
]. E3 is a homodimer with two identical active sites localized at the interface between two subunits. E3 monomer has four structural domains: FAD-binding domain, NAD+
-binding domain, the central domain and the interface domain. Two tightly bound FAD molecules per dimeric E3 are involved in the electron transfer from dihydrolipoamide to NAD+
with participation of the disulfide of the active site. In hE3 catalysis involves H452 acting as an active-site weak base, E457 stabilizing H452, and P453 positioning H452 close to the redox disulfide [11
]. Recently the structures of hE3 with bound NAD+
and with bound NADH [13
] and as a subcomplex with the E3-binding domain of hBP were determined [14
]. Binding of hE3 to the E3-binding domain of hBP did not cause any detectable conformational changes in the hE3 structure. The structures indicated electrostatic as well as hydrophobic interactions between two proteins [14
We have examined the binding regions of the hE1 to the E1-binding domain of hE2 and hE3 and the E3-binding domain of hBP. Our findings confirm the role of several amino acid residues of hBP involved in binding based on the 3D structure and provide insights in the binding of hE1 and the E1-binding domain of hE2 even though the 3D structure of this subcomplex remains unknown.