Domain structures of the dihydrolipoyl transacetylase and the protein X components of mammalian pyruvate dehydrogenase complex. Selective cleavage by protease Arg C

Treatment of the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2-X-KcKb) with protease Arg C selectively converted protein X into an inner domain fragment (Mr approximately equal to 35,000) and an outer (lipoyl-bearing) domain fragment (Mr approximately equal to 15,500). These fragments were larger and much smaller, respectively, than the inner domain and outer domain fragments derived from the E2 component, supporting the conclusion that protein X is distinct from the E2 component. Protease Arg C cleaved the Kb subunit more slowly than protein X. An increase in kinase activity correlated with this cleavage of the Kb subunits. An even slower cleavage of E2 subunits generated an inner domain fragment (Mr approximately equal to 31,500) and a lipoyl-bearing domain fragment (Mr approximately equal to 49,000) which had Mr values at least 3,000 and 10,000 larger, respectively, than the corresponding E2 fragments generated by trypsin treatment of the subcomplex. Following various extents of cleavage with protease Arg C or trypsin, residual oligomeric subcomplexes were isolated and characterized. We found that selective removal of the lipoyl-bearing domain of protein X did not alter lipoyl-mediated regulation of the kinase indicating that the lipoyl residues bound to E2 subunits are effective, that the inner domain of protein X remained associated with the inner domain of E2 subunits following the complete removal of the outer domains of both E2 and protein X, that, with only 10% of the E2 subunits intact, nearly half of the catalytic (Kc) subunits of the kinase were bound by the residual subcomplex, and that removal of the remaining outer domains from E2 subunits released the Kc subunits. Thus, protein X is unique among the subunits of the complex in binding tightly to the oligomeric inner domain of the transacetylase, and the outer domain of the transacetylase serves to bind to and facilitate the regulation of the catalytic subunit of the kinase.     

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.     

Additional binding sites for the pyruvate dehydrogenase kinase but not for protein X in the assembled core of the mammalian pyruvate dehydrogenase complex: binding region for the kinase.

A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of approximately 60 dihydrolipoyl transacetylase (E2) subunits, approximately 6 protein X subunits, and approximately 2 pyruvate dehydrogenase kinase heterodimers (KcKb). Using a preparation of resolved kinase in which Kc much greater than Kb, E2-X-KcKb subcomplex additionally bound at least 15 catalytic subunits of the kinase (Kc) and a much lower level of Kb. The binding of Kc to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-KcKb subcomplex. This pattern of binding Kc but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the Kc subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the kinase (M. Rahmatullah et al., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugation yielded an E2L-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2.     

Component requirements for NADH inhibition and spermine stimulation of pyruvate dehydrogenaseb phosphatase activity

The subunit and subdomain requirements for NADH inhibition as well as Ca+ and spermine activation of the pyruvate dehydrogenaseb phosphatase were analyzed. The transacetylase-protein X subcomplex (E2-X) was required for all three effects. The oligomeric inner domain of the transacetylase did not support any of these regulatory effects. The presence of at least a portion of the outer (lipoyl-bearing) domains of the transacetylase but not the lipoyl-bearing portion of protein X was essential for expression of these regulatory effects on phosphatase activity. The inner domain of protein X may contribute to some effects. The E2-X subcomplex, alone, had no effect on phosphatase activity in the absence of Ca2+, but the subcomplex did support both NADH inhibition and spermine activation in the absence of Ca2+. Studies with peptide substrates established that spermine is directly bound by a phosphatase subunit. With the resolved pyruvate dehydrogenase component (E1b) used as the substrate, the E2-X subcomplex transformed the effect of spermine from inhibiting to stimulating the rate of dephosphorylation by the phosphatase. The above observations suggest that binding of E1b to the E2-X subcomplex alters its presentation to the phosphatase. We also present several observations that are consistent with NADH inhibition of the phosphatase being mediated through a dihydrolipoyl dehydrogenase-dependent reduction of lipoyl moieties in the E2-X subcomplex. Overall, our data establish that the outer, lipoyl-bearing domains of the oligomeric transacetylase core have an essential role in the function and regulation of the pyruvate dehydrogenase phosphatase.     

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.     

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.     

Separation of protein X from the dihydrolipoyl transacetylase component of the mammalian pyruvate dehydrogenase complex and function of protein X

The dihydrolipoyl transacetylase (E2)-protein X-kinase subcomplex was resolved to produce an oligomeric transacetylase that was free of protein X and kinase subunits. We investigated the properties of this transacetylase E2 oligomer and of a form of the subcomplex from which only the lipoyl-bearing domain of protein X (XL) was removed. While retaining other catalytic and binding properties of the native subcomplex, the oligomeric transacetylase and the subcomplex lacking the XL domain had greatly reduced capacities both to support the overall reaction of the complex (upon reconstitution with other components) and to bind the dihydrolipoyl dehydrogenase component. Our results indicate that protein X, in part through its XL domain, contributes to the binding of the dihydrolipoyl dehydrogenase component and to the overall reaction of the complex.     

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.     

Marked differences between two isoforms of human pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.     

Essential roles of lipoyl domains in the activated function and control of pyruvate dehydrogenase kinases and phosphatase isoform 1.

Four pyruvate dehydrogenase kinase and two pyruvate dehydrogenase phosphatase isoforms function in adjusting the activation state of the pyruvate dehydrogenase complex (PDC) through determining the fraction of active (nonphosphorylated) pyruvate dehydrogenase component. Necessary adaptations of PDC activity with varying metabolic requirements in different tissues and cell types are met by the selective expression and pronounced variation in the inherent functional properties and effector sensitivities of these regulatory enzymes. This review emphasizes how the foremost changes in the kinase and phosphatase activities issue from the dynamic, effector-modified interactions of these regulatory enzymes with the flexibly held outer domains of the core-forming dihydrolipoyl acetyl transferase component.     

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).     

Pyruvate dehydrogenase kinase isoform 2 activity stimulated by speeding up the rate of dissociation of ADP

Pyruvate dehydrogenase kinase 2 (PDK2) activity is stimulated by NADH and NADH plus acetyl-CoA via the reduction and reductive acetylation of the lipoyl groups of the dihydrolipoyl acetyltransferase (E2) component. Elevated K(+) and Cl(-) were needed for significant stimulation. Stimulation substantially increased both k(cat) and the K(m) for ATP; the fractional stimulation increased with the level of ATP. With an E2 structure lacking the pyruvate dehydrogenase (E1) binding domain, stimulation of PDK2 was retained, the K(m) for E1 decreased, and the equilibrium dissociation constant for ATP increased but remained much lower than the K(m) for ATP. Stimulation of PDK2 activity greatly reduced the fraction of bound ADP. These results fit an ordered reaction mechanism with ATP binding before E1 and stimulation increasing the rate of dissociation of ADP. Conversion of all of the lipoyl groups in the E2 60mer to the oxidized form (E2(ox)) greatly reduced k(cat) and the K(m) of PDK2 for ATP. Retention over an extended period of time of a low portion of reduced lipoyl groups maintains E2 in a state that supported much higher PDK2 activity than short-term (5 min) reduction of a large portion of lipoyl groups of E2(ox), but reduction of E2(ox) produced a larger fold stimulation. Reduction and to a greater extent reductive acetylation increased PDK2 binding to E2; conversion to E2(ox) did not significantly hinder binding. We suggest that passing even limited reducing equivalents among lipoyl groups maintains E2 lipoyl domains in a conformation that aids kinase function.     

Conservation of primary structure in the lipoyl-bearing and dihydrolipoyl dehydrogenase binding domains of mammalian branched-chain alpha-keto acid dehydrogenase complex: molecular cloning of human and bovine transacylase (E2) cDNAs

The subunit structures and conservation of the dihydrolipoyl transacylase (E2) components of bovine and human branched-chain alpha-keto acid dehydrogenase complexes were investigated by Western blotting, peptide sequencing, and cDNA cloning methods. Rabbit antiserum prepared against the sodium dodecyl sulfate (SDS) denaturated bovine E2 subunit recognized the inner E2 core, and the first hinge region of the E2 chain, but failed to react with the lipoyl-bearing domain as determined by Western blot analysis. The lack of antigenicity in the lipoyl-bearing domain was confirmed with antibodies directed against the native E2 component. A human E2 cDNA (1.6 kb) was isolated from a human liver cDNA library in lambda gt11 with a combination of the above anti-native and anti-SDS-denatured E2 immunoglobulin G's as a probe. The fidelity of the human E2 cDNA was established by nucleotide sequencing which showed the determined peptide sequences of the amino terminus and tryptic fragments of bovine E2. A bovine E2 cDNA (0.7 kb) was also isolated from a bovine liver cDNA library in lambda ZAP with the human E2 cDNA as a probe. Northern blot analysis using the human E2 cDNA probe showed that E2 mRNAs in bovine liver and human kidney mesangial cells are 3.3 and 4.6 kb in size, respectively. Primary structures derived from human and bovine E2 cDNAs show leader sequences including the initiator methionine and the homologous mature peptides consisting of complete lipoyl-bearing and dihydrolipoyl dehydrogenase (E3) binding domains and two hinge regions. In addition, the human E2 cDNA contains a portion of the inner E2 core sequence, a 3'-untranslated region, and a poly(A+) tail. Deduced amino acid sequences of the mammalian E2's were compared with those of Escherichia coli transacetylase and transsuccinylase and bovine kidney transacetylase. The results indicate a high degree of conservation in the sequence flanking the lipoyl-attachment site and in the E3-binding domain. Models are presented to discuss implications for the conserved structure-function relationship in the lipoyl-bearing and E3-binding domains of alpha-keto acid dehydrogenase complexes.     

Component X. An immunologically distinct polypeptide associated with mammalian pyruvate dehydrogenase multi-enzyme complex

The mammalian pyruvate dehydrogenase multi-enzyme complex contains a tightly-associated 50 000-Mr polypeptide of unknown function (component X) in addition to its three constituent enzymes, pyruvate dehydrogenase (E1), lipoate acetyltransferase (E2) and lipoamide dehydrogenase (E3) which are jointly responsible for production of CoASAc and NADH. The presence of component X is apparent on sodium dodecyl sulphate/polyacrylamide gel analysis of the complex, performed in Tris-glycine buffers although it co-migrates with the E3 subunit on standard phosphate gels run under denaturing conditions. Refined immunological techniques, employing subunit-specific antisera to individual components of the pyruvate dehydrogenase complex, have demonstrated that protein X is not a proteolytic fragment of E2 (or E3) as suggested previously. In addition, anti-X serum elicits no cross-reaction with either subunit of the intrinsic kinase of the pyruvate dehydrogenase complex. Immune-blotting analysis of SDS extracts of bovine, rat and pig cell lines and derived subcellular fractions have indicated that protein X is a normal cellular component with a specific mitochondrial location. It remains tightly-associated with the 'core' enzyme, E2, on dissociation of the complex at pH 9.5 or by treatment with 0.25 M MgCl2. This polypeptide is not released to any significant extent from E2 by p-hydroxymercuriphenyl sulphonate, a reagent which promotes dissociation of the specific kinase of the complex from the 'core' enzyme. Incubation of the complex with [2-14C]pyruvate in the absence of CoASH promotes the incorporation of radio-label, probably in the form of acetyl groups, into both E2 and component X.     

Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex

The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.     

The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity

Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoyl-bearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoyl-bearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes.     

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.     

Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP

Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating.     

Recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2

Pyruvate dehydrogenase kinase 2 (PDHK2) is a unique mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase multienzyme complex (PDC). PDHK2 is an integral component of PDC tightly bound to the inner lipoyl-bearing domains (L2) of the dihydrolipoyl transacetylase component (E2) of PDC. This association has been reported to bring about an up to 10-fold increase in kinase activity. Despite the central role played by E2 in the maintenance of PDHK2 functionality in the PDC-bound state, the molecular mechanisms responsible for the recognition of L2 by PDHK2 and for the E2-dependent PDHK2 activation are largely unknown. In this study, we used a combination of molecular modeling and site-directed mutagenesis to identify the amino acid residues essential for the interaction between PDHK2 and L2 and for the activation of PDHK2 by E2. On the basis of the results of site-directed mutagenesis, it appears that a number of PDHK2 residues located in its R domain (P22, L23, F28, F31, F44, L45, and L160) and in the so-called "cross arm" structure (K368, R372, and K391) are critical in determining the strength of the interaction between PDHK2 and L2. The residues of L2 essential for recognition by PDHK2 include L140, K173, I176, E179, and to a lesser extent D164, D172, and A174. Importantly, certain PDHK2 residues forming interfaces with L2, i.e., K17, P22, F31, F44, R372, and K391, are also critical for the maintenance of enhanced PDHK2 activity in the E2-bound state. Finally, evidence that the blood glucose-lowering compound AZD7545 disrupts the interactions between PDHK2 and L2 and thereby inhibits PDHK2 activity is presented.     

Cryoelectron microscopy of mammalian pyruvate dehydrogenase complex.

Cryoelectron microscopy has been performed on frozen-hydrated pyruvate dehydrogenase complexes from bovine heart and kidney and on various subcomplexes consisting of the dihydrolipoyl transacetylase-based (E2) core and substoichiometric levels of the other two major components, pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3). The diameter of frozen-hydrated pyruvate dehydrogenase complex (PDC) is 50 nm, which is significantly larger than previously reported values. On the basis of micrographs of the subcomplexes, it is concluded that the E1 and E3 are attached to the E2-core complex by extended (4-6 nm maximally) flexible tethers. PDC constructed in this manner would probably collapse and appear smaller than its native size when dehydrated, as was the case in previous electron microscopy studies. The tether linking E1 to the core involves the hinge sequence located between the E1-binding and catalytic domains in the primary sequence of E2, whereas the tether linking E3 is probably derived from a similar hinge-type sequence in component X. Tilting of the E2-based cores and comparison with model structures confirmed that their overall shape is that of a pentagonal dodecahedron. The approximately 6 copies of protein X present in PDC do not appear to be clustered in one or two regions of the complex and are not likely to be symmetrically distributed.