Cryoelectron microscopy of frozen-hydrated alpha-ketoacid dehydrogenase complexes from Escherichia coli

The native architectures of the pyruvate and 2-oxoglutarate dehydrogenase complexes have been investigated by cryoelectron microscopy of unstained, frozen-hydrated specimens. In pyruvate dehydrogenase complex and 2-oxoglutarate dehydrogenase complex the transacylase (E2) components exist as 24-subunit, cube-shaped assemblies that form the structural cores of the complexes. Multiple copies (12-24) of the alpha-ketoacid dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) components bind to the surface of the cores. Images of the frozen-hydrated enzyme complexes do not appear consistent with a symmetric arrangement of the E1 and E3 subunits about the octahedrally symmetric E2 core. Often the E1 or E3 subunits appear separated from the surface of the E2 core by 3-5 nm, and sometimes thin bridges of density appear in the gap between the E2 core and the bound subunits; studies of subcomplexes consisting of the E2 core from 2-oxoglutarate dehydrogenase complex and E1 or E3 show that both E1 and E3 are bound in this manner. Images of the E2 cores isolated from pyruvate dehydrogenase complex appear surrounded by a faint fuzz that extends approximately 10 nm from the surface of the core and likely corresponds to the lipoyl domains of the E2.     

Configuration of interdomain linkers in pyruvate dehydrogenase complex of Escherichia coli as determined by cryoelectron microscopy.

The dihydrolipoyl transacetylase (E2p) component of the pyruvate dehydrogenase complex (PDC) of Escherichia coli is a multidomain polypeptide comprising a catalytic domain, a domain that binds dihydrolipoyl dehydrogenase (E3-binding domain), and three domains containing lipoic acid (lipoyl domains). In PDC 24 subunits of E2p associate by means of interactions involving the catalytic domains to form the structural core of PDC. From cryoelectron microscopy and computer image analysis of frozen-hydrated isolated E2p cores it appears that the lipoyl domains are located peripherally about the core complex and do not assume fixed positions. To further test this interpretation the visibility of the lipoyl domains in electron micrographs was enhanced by specifically biotinylating the lipoic acids and labeling them with streptavidin. In agreement with the studies of native, unlabeled E2p cores, cryoelectron microscopy of the streptavidin-labeled E2p cores showed that the lipoic acid moieties are capable of extending approximately 13 nm from the surface of the core. Localization of the E3-binding domains was accomplished by cryoelectron microscopy of E2p-E3 subcomplexes prepared by reconstitution in vitro. Frequently an apparent gap of several nanometers separated the bound E3 from the surface of the core. The third component of PDC, pyruvate dehydrogenase (E1p), appeared to bind to the E2p core in a manner similar to that observed for E3. These results support a structural model of the E2p core in which the catalytic, E3-binding, and three lipoyl domains are interconnected by linker sequences that assume extended and flexible conformations.     

Regulation of pyruvate dehydrogenase complex activity in Ehrlich ascites carcinoma cells by Ca2+ and pyruvate

The dependence of pyruvate dehydrogenase complex (PDC) activity on [Ca2+] was determined in Ehrlich ascites carcinoma cells at different pyruvate concentrations. The resulting family of curves had the following characteristics: a) bell-shaped appearance of all curves with maximum activity at 600 nM Ca2+; b) unchanged position of maxima with changes in pyruvate concentration; c) nonmonotonous changes in PDC activity with increasing pyruvate concentration at fixed [Ca2+]. Feasible mechanisms involving Ca2+-dependent phosphatase and kinase which are consistent with the experimental findings are discussed. To determine the steps in the chain of PDC reactions which determine the observed phenomena, a mathematical model is suggested which is based on the known data on the structural--functional relationships between the complex components--pyruvate dehydrogenase (E1), dihydrolipoyl acetyl transferase (E2), dihydrolipoyl dehydrogenase (E3), protein X, kinase, and phosphatase. To adequately describe the non-trivial dependence of PDC activity on [Ca2+] at different pyruvate concentrations, it was also necessary to consider the interdependence of some steps in the general chain of PDC reactions. Phenomenon (a) is shown to be due only to the involvement of protein X in the PDC reactions, phenomenon (b) to be due to changes in the activity of kinase, and phenomenon (c) to be due to dependence of acetylation and transacetylation rates on pyruvate concentration.     

Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775.

In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram-positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1 alpha and E1 beta), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram-positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions.     

The pyruvate dehydrogenase complex from the parasitic nematode Ascaris suum: novel subunit composition and domain structure of the dihydrolipoyl transacetylase component.

The pyruvate dehydrogenase complex (PDC) from muscle of the adult parasitic nematode Ascaris suum plays a unique role in its anaerobic mitochondrial metabolism. Resolution of the intact complex in high salt dissociates the pyruvate dehydrogenase subunit but leaves the dihydrolipoyl dehydrogenase subunit (E3) and two other proteins with apparent M(r)s of 45 and 43 kDa bound to the dihydrolipoyl transacetylase (E2) core. These proteins are not observable on Coomassie brilliant blue-stained gels of other eukaryotic PDCs, but the 45-kDa protein is similar in apparent M(r), pI, and sensitivity to trypsin to the Kb subunit of the bovine kidney PDH alpha kinase. Acetylation of the ascarid PDC with [2-14C]pyruvate under conditions designed to maximize the incorporation of label into protein yielded only a single radiolabeled subunit, E2. These results confirm earlier reports that the ascarid PDC lacks protein X, an integral component recently identified in other eukaryotic PDCs. About 1.6 to 1.8 mol of 14C was incorporated/mole of E2, suggesting that the ascarid E2 contained two lipoly-bearing domains. Domain mapping of the 14C-acetylated ascarid E2 by limited tryptic digestion identified two lipoyl-bearing fragments with apparent M(r)s of 50 and 34 kDa and two core fragments with apparent M(r)s of 46 and 30 kDa. The ascarid E2 domain structure appears to be similar to that of other E2s. However, it appears that the subunit-binding domain (E2B) of the ascarid E2 may be significantly larger or be flanked by larger than normal interdomain regions. An enlarged E2B domain may be necessary to accommodate the additional binding of E3 to the E2 subunit in the ascarid complex, in the absence of protein X.     

A new level of architectural complexity in the human pyruvate dehydrogenase complex

Mammalian pyruvate dehydrogenase multienzyme complex (PDC) is a key metabolic assembly comprising a 60-meric pentagonal dodecahedral E2 (dihydrolipoamide acetyltransferase) core attached to which are 30 pyruvate decarboxylase E1 heterotetramers and 6 dihydrolipoamide dehydrogenase E3 homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3-binding protein (E3BP) located on each of the 12 E2 icosahedral faces. Here, we present evidence for a novel subunit organization in which E3 and E3BP form subcomplexes with a 1:2 stoichiometry implying the existence of a network of E3 "cross-bridges" linking pairs of E3BPs across the surface of the E2 core assembly. We have also determined a low resolution structure for a truncated E3BP/E3 subcomplex using small angle x-ray scattering showing one of the E3BP lipoyl domains docked into the E3 active site. This new level of architectural complexity in mammalian PDC contrasts with the recently published crystal structure of human E3 complexed with its cognate subunit binding domain and provides important new insights into subunit organization, its catalytic mechanism and regulation by the intrinsic PDC kinase.     

Detailed evaluation of pyruvate dehydrogenase complex inhibition in simulated exercise conditions

The mammalian pyruvate dehydrogenase complex (PDC) is a mitochondrial multienzyme complex that connects glycolysis to the tricarboxylic acid cycle by catalyzing pyruvate oxidation to produce acetyl-CoA, NADH, and CO₂. This reaction is required to aerobically utilize glucose, a preferred metabolic fuel, and is composed of three core enzymes: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). The pyruvate-dehydrogenase-specific kinase (PDK) and pyruvate-dehydrogenase-specific phosphatase (PDP) are considered the main control mechanism of mammalian PDC activity. However, PDK and PDP activity are allosterically regulated by several effectors fully overlapping PDC substrates and products. This collection of positive and negative feedback mechanisms confounds simple predictions of relative PDC flux, especially when all effectors are dynamically modulated during metabolic states that exist in physiologically realistic conditions, such as exercise. Here, we provide, to our knowledge, the first globally fitted, pH-dependent kinetic model of the PDC accounting for the PDC core reaction because it is regulated by PDK, PDP, metal binding equilibria, and numerous allosteric effectors. The model was used to compute PDH regulatory complex flux as a function of previously determined metabolic conditions used to simulate exercise and demonstrates increased flux with exercise. Our model reveals that PDC flux in physiological conditions is primarily inhibited by product inhibition (～60%), mostly NADH inhibition (～30–50%), rather than phosphorylation cycle inhibition (～40%), but the degree to which depends on the metabolic state and PDC tissue source.     

Escherichia coli pyruvate dehydrogenase complex: particle masses of the complex and component enzymes measured by scanning transmission electron microscopy

Particle masses of the Escherichia coli pyruvate dehydrogenase (PDH) complex and its component enzymes have been measured by scanning transmission electron microscopy (STEM). The particle mass of PDH complex measured by STEM is 5.28 X 10(6) with a standard deviation of 0.40 X 10(6). The masses of the component enzymes together with their standard deviations are (2.06 +/- 0.26) X 10(5) for the dimeric pyruvate dehydrogenase (E1), (1.15 +/- 0.17) X 10(5) for dimeric dihydrolipoyl dehydrogenase (E3), and (2.20 +/- 0.17) X 10(6) for dihydrolipoyl transacetylase (E2), the 24-subunit core enzyme. The latter value corresponds to a subunit molecular weight of (9.17 +/- 0.71) X 10(4) for E2. The subunit molecular weight measured by polyacrylamide gel electrophoresis in sodium dodecyl sulfate is 8.6 X 10(4). STEM measurements on PDH complex incubated with excess E3 or E1 failed to detect any additional binding of E3 but showed that the complex would bind additional E1 under forcing conditions (high concentrations with glutaraldehyde). The additional E1 subunits were bound too weakly to represent binding sites in an isolated or isolable complex. The mass measurements by STEM are consistent with the subunit composition 24:24:12 when interpreted in the light of the flavin content of the complex and assuming 24 subunits in the core enzyme (E2).     

Scanning transmission electron microscopic study of alpha-ketoglutarate dehydrogenase complex from Escherichia coli

The alpha-ketoglutarate dehydrogenase complex was resolved into its three component enzymes: alpha-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase. Subcomplexes were prepared in vitro by incubating the resolved E2, a 24-subunit cube-shaped molecule, with E1 (dimeric). The morphology and mass of the subcomplexes were determined by scanning transmission electron microscopy of negatively stained and of freeze-dried specimens. Images of both negative stained and freeze-dried subcomplexes were consistent with E1 binding at or near the midpoints of the edges of the E2 molecule. Mass analysis of the freeze-dried specimen showed that at least 95% of E1 remains in the dimeric state (or as two closely juxtaposed monomers) when it binds to E2.     

Self-assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus.

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, alpha2beta2) and dihydrolipoyl dehydrogenase (E3, alpha2) 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.     

Component X of mammalian pyruvate dehydrogenase complex: structural and functional relationship to the lipoate acetyltransferase (E2) component

The lipoate acetyltransferase (E2, Mr 70,000) and protein X (Mr 51,000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N-terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C-labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly-arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N-terminus.     

The quaternary structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. A reconsideration

After limited proteolysis of the dihydrolipoyl transacetylase component (E2) of Azotobacter vinelandii pyruvate dehydrogenase complex (PDC), a C-terminal domain was obtained which retained the transacetylase active site and the quaternary structure of E2 but had lost the lipoyl-containing N-terminal part of the chain and the binding sites for the peripheral components, pyruvate dehydrogenase and lipoamide dehydrogenase. The C-terminus of this domain was determined by treatment with carboxypeptidase Y and shown to be identical with the C-terminus of E2. Together with the previously determined N-terminus and the known amino acid sequence of E2, a molecular mass of 27.5 kDa was calculated. From the molecular mass of the native catalytic domain, 530 kDa, and the symmetry of the cubic structures observed on electron micrographs, a 24-meric structure is concluded instead of the 32-meric structure proposed previously. From the effect of guanidine hydrochloride on the light-scattering of intact E2 it was concluded that dissociation occurs in a two-step reaction resulting in particles with an average mass 1/6 that of the original mass before the N----D transition takes place. Cross-linking experiments with the catalytic domain indicated that the multimeric E2 is built from tetramers and that the tetramers are arranged as a dimer of dimers. A model for the quaternary structure of E2 is given, in which it is assumed that the tetrameric E2 core of PDC is formed from each of the six morphological subunits located at the lateral face of the cube. Binding of peripheral components to a site that interferes with the cubic assembly causes dissociation, resulting in the unique small PDC of A. vinelandii.     

Properties of the pyruvate dehydrogenase kinase bound to and separated from the dihydrolipoyl transacetylase-protein X subcomplex and evidence for binding of the kinase to protein X

Studies were conducted on four pyruvate dehydrogenase kinase-containing fractions: purified pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2.X.K), a kinase fraction (K fraction) prepared from the E2.X.K subcomplex, and a kinase fraction generated by limited trypsin-digestion of E2.X.K. We characterized the gel electrophoresis properties of dissociated subunits (one-dimensional and two-dimensional), the catalytic and ATP binding properties of kinase-containing fractions, and the subunit requirements for kinase binding to and being activated by the transacetylase-protein X subcomplex (E2.X). A significant portion of protein X was retained with the transacetylase core following release of virtually all the kinase. The K fraction had four major bands separated by sodium dodecyl sulfate-slab gel electrophoresis which corresponded to the dihydrolipoyl dehydrogenase, protein X, the trypsin-resistant catalytic subunit of the kinase and a chymotrypsin-resistant subunit which had a high pI and comigrated in one-dimensional systems with the chymotrypsin-sensitive alpha-subunit of the pyruvate dehydrogenase component. While purified kidney complex contained only about three molecules of kinase (determined by [14C]ATP binding), one molecule of E2.X subcomplex activated a large number (greater than 15) molecules of kinase associated with the protein X-containing K fraction. Sephadex G-200 chromatography of the K fraction in the presence of dithiothreitol led to coelution of protein X and kinase subunits. Limited trypsin digestion converted the transacetylase into subdomains and cleaved protein X and the high pI subunit of the kinase. Under those conditions, the intact catalytic subunit of the kinase did not bind to the large inner domain of the transacetylase but could be activated by untreated E2.X subcomplex. Thus, binding of the catalytic subunit of the kinase and its activation by E2.X required either protein X or the lipoyl-bearing outer domain of the transacetylase. In combination, our results suggest that protein X serves to anchor the kinase to the core of the complex.     

A model for the spatial location of pyruvate dehydrogenase phosphatase in mammalian pyruvate dehydrogenase complex

Recent experimental findings on the structural--functional features of pyruvate dehydrogenase phosphatase (PDP) isolated from various sources are compared. Two alternative mechanisms (a and b) of dephosphorylation of the E1 component in the pyruvate dehydrogenase complex (PDC) are discussed: a) the reaction occurs as a result of stochastic collisions of PDP and PDC, and the generation of an enzyme--substrate complex (PDP--E1--PDC) and dephosphorylation of the E1 component occur independently at different PDP binding sites on the PDC core; b) the dephosphorylation is performed simultaneously by a certain number of PDP molecules symmetrically bound on the PDC core. The second mechanism is suggested by the self-assembly theory of multicomponent enzyme systems and can be proved by kinetic experiments. Based on self-assembly principles and data on feasible binding sites of peripheral components of the PDC, the stoichiometry and mutual location of PDP, pyruvate dehydrogenase kinase, and the E1 component on the core of mammalian PDC are postulated to provide optimal functioning of the PDC. Structural mechanisms of stimulation of PDP activity by Ca2+ and polyamines are also discussed.     

Crystallization of a dihydrolipoyl transacetylase--dihydrolipoyl dehydrogenase subcomplex and its implications regarding the subunit structure of the pyruvate dehydrogenase complex from Escherichia coli.

A subcomplex consisting of dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase, two of the three enzymes comprising the Escherichia coli pyruvate dehydrogenase complex, has been crystallized. X-ray diffraction data establish that the space group is P2₁3 with unit cell dimension $\text{a=211 .5}\text{A}\text{?}$. The unit cell contains four molecules of the subcomplex, each possessing 3-fold crystallographic and molecular symmetry. This finding, together with biochemical and electron microscopic data reported elsewhere, establish unequivocally that dihydrolipoyl transacetylase, the core enzyme of the pyruvate dehydrogenase complex, consists of 24 identical subunits with octahedral (432) symmetry. In the case presented here, the 432 symmetry of the transacetylase is reduced to 3-fold symmetry in the subcomplex by the addition of dihydrolipoyl dehydrogenase subunits. Crystal density measurements indicate that the dihydrolipoyl transacetylase present in these crystals is considerably smaller than the core mass generally reported for intact transacetylase. The implications of these findings are discussed with respect to the subunit stoichiometry and structure of the E. coli pyruvate dehydrogenase complex.     

Properties of a newly characterized protein of the bovine kidney pyruvate dehydrogenase complex

The dihydrolipoyl transacetylase component, which serves as the structural core of mammalian pyruvate dehydrogenase complexes, is acetylated when treated with either pyruvate or with acetyl-CoA in the presence of NADH. Besides the dihydrolipoyl transacetylase component, we have found that another protein, referred to as protein X, is rapidly acetylated at thiol residues. Protein X remains fully bound to the transacetylase core under conditions that remove the pyruvate dehydrogenase and dihydrolipoyl dehydrogenase components. Mapping of 125I-tryptic peptides indicated that the transacetylase subunits and protein X are structurally distinct; however, under the same mapping conditions, there is considerable similarity in the positions of acetylated peptides derived from these subunits. Affinity-purified rabbit immunoglobulin G prepared against the dihydrolipoyl transacetylase core reacted exclusively with the transacetylase and with both its tryptic-derived inner domain and outer lipolyl-bearing domain. Those results further indicate that protein X is not derived from the transacetylase subunit Affinity-purified mouse antibody to protein X reacted selectively with large tryptic polypeptides derived from protein X and did not react with the inner domain of the transacetylase. However, the anti-protein X antibody did react with the intact transacetylase subunit, the lipoyl-bearing domain of the transacetylase, and weakly with the transsuccinylase component of the alpha-ketoglutarate dehydrogenase complex. This cross-reactivity reflected specificity of a portion of the polyclonal antibodies for a related structural region in the transacetylase and protein X (possibly a similar lipoyl-bearing region). Furthermore, a major portion of that polyclonal antibody was shown to react exclusively with protein X. Thus, protein X subunits differ substantially from transacetylase subunits but the two components have a region of structural similarity. We estimate that there are about 5 mol of protein X per mol of the kidney pyruvate dehydrogenase complex. Under a variety of conditions that result in a wide range of levels of acetylation of sites in the complex, about 1 acetyl group is incorporated into protein X per 10 acetyl groups incorporated into the transacetylase subunits per mol of complex. That ratio is close to the ratio of protein X subunits of transacetylase subunits in the complex, indicating that there are efficient mechanisms for acylation and deacylation of protein X.     

The catalytic requirements for reduction and acetylation of protein X and the related regulation of various forms of resolved pyruvate dehydrogenase kinase

The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core component (E2) by another subunit, referred to as protein X (Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261, 6515-6523). We determined the catalytic requirements for reduction and acetylation of the lipoyl moiety in protein X and linked those changes in protein X to regulatory effects on kinase activity. Using fractions prepared by resolution and proteolytic treatments, we evaluated which subunits are required for regulatory effects on kinase activity. With X-KcKb fraction (treated to remove the mercurial agent used in its preparation), we found that the resolved pyruvate dehydrogenase component, the isolated inner domain of E2 (lacking the lipoyl-bearing region of E2), and the dihydrolipoyl dehydrogenase component directly utilize protein X as a substrate. The resulting reduction and acetylation of protein X occurs in association with enhancement of kinase activity. Following tryptic cleavage of E2 and protein X into subdomains, full acetylation of the lipoyl-bearing subdomains of these proteins is retained along with the capacity of acetylating substrates to stimulate kinase activity. All kinase-containing fractions, including those in which the Kb subunit was digested, were inhibited by pyruvate or ADP, alone, and synergistically by the combination suggesting that pyruvate and ADP bind to Kc. Our results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects. A dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.     

Crystal structure of pyruvate dehydrogenase phosphatase 1 and its functional implications

Pyruvate dehydrogenase phosphatase 1 (PDP1) catalyzes dephosphorylation of pyruvate dehydrogenase (E1) in the mammalian pyruvate dehydrogenase complex (PDC), whose activity is regulated by the phosphorylation-dephosphorylation cycle by the corresponding protein kinases (PDHKs) and phosphatases. The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2 (inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. Here, we report the crystal structure of the rat PDP1c at 1.8 A resolution. The structure reveals that PDP1 belongs to the PPM family of protein serine/threonine phosphatases, which, in spite of a low level of sequence identity, share the structural core consisting of the central beta-sandwich flanked on both sides by loops and alpha-helices. Consistent with the previous studies, two well-fixed magnesium ions are coordinated by five active site residues and five water molecules in the PDP1c catalytic center. Structural analysis indicates that, while the central portion of the PDP1c molecule is highly conserved among the members of the PPM protein family, a number of structural insertions and deletions located at the periphery of PDP1c likely define its functional specificity towards the PDC. One notable feature of PDP1c is a long insertion (residues 98-151) forming a unique hydrophobic pocket on the surface that likely accommodates the lipoyl moiety of the E2 domain in a fashion similar to that of PDHKs. The cavity, however, appears more open than in PDHK, suggesting that its closure may be required to achieve tight, specific binding of the lipoic acid. We propose a mechanism in which the closure of the lipoic acid binding site is triggered by the formation of the intermolecular (PDP1c/L2) Ca2+ binding site in a manner reminiscent of the Ca2+ -induced closure of the regulatory domain of troponin C.     

Molecular structure of a 9-MDa icosahedral pyruvate dehydrogenase subcomplex containing the E2 and E3 enzymes using cryoelectron microscopy

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (alpha(2)beta(2)) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of approximately 90 A separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of approximately 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.     

Reaction mechanism for mammalian pyruvate dehydrogenase using natural lipoyl domain substrates

The pyruvate dehydrogenase (E1) component of the pyruvate dehydrogenase complex (PDC) catalyzes a two-step reaction. Recombinant production of substrate amounts of the lipoyl domains of the dihydrolipoyl transacetylase (E2) component of the mammalian PDC allowed kinetic characterization of the rapid physiological reaction catalyzed by E1. Using either the N-terminal (L1) or the internal (L2) lipoyl domain of E2 as a substrate, analyses of steady state kinetic data support a ping pong mechanism. Using standard E1 preparations, Michaelis constants (Km) were 52 +/- 14 microM for L1 and 24.8 +/- 3.8 microM for pyruvate and k(cat) was 26.3 s(-1). With less common, higher activity preparations of E1, the Km values were > or =160 microM for L1 and > or =35 microM for pyruvate and k(cat) was > or =70 s(-1). Similar results were found with the L2 domain. The best synthetic lipoylated-peptide (L2 residues 163-177) was a much poorer substrate (Km > or =15 mM, k(cat) approximately equals 5 s(-1); k(cat)/Km decreased >1,500-fold) than L1 or L2, but a far better substrate in the E1 reaction than free lipoamide (k(cat)/Km increased >500-fold). Each lipoate source was an effective substrate in the dihydrolipoyl dehydrogenase (E3) reaction, but E3 had a lower Km for the L2 domain than for lipoamide or the lipoylated peptides. In contrast to measurements with slow E1 model reactions that use artificial acceptors, we confirmed that the natural E1 reaction, using lipoyl domain acceptors, was completely inhibited (>99%) by phosphorylation of E1 and the phosphorylation strongly inhibited the reverse of the second step catalyzed by E1. The mechanisms by which phosphorylation interferes with E1 activity is interpreted based on accrued results and the location of phosphorylation sites mapped onto the 3-D structure of related alpha-keto acid dehydrogenases.