Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The intramolecular passage of substrate between the component enzymes of the pyruvate dehydrogenase multienzyme complex of Escherichia coli was examined. A series of partly reassembled complexes, varying only in their E1 (pyruvate decarboxylase, EC 1.2.4.1) content, was incubated with pyruvate in the absence of CoA, conditions under which the lipoic acid residues covalently bound to the E2 (lipoate acetyltransferase, EC2.3.1.12) chains of the complex become reductively acetylated, and the reaction then ceases. The fraction of E2 chains thus acetylated was estimated by specific reaction of the thiol groups in the acetyl-lipoic acid moieties with N-ethyl[2,3-14C]maleimide. The simplest interpretation of the results was that a single E1 dimer is capable of catalysing the rapid acetylation of 8-12 E2 chains, in good agreement with the results of Bates, Danson, Hale, Hooper & Perham [(1977) Nature (London) 268, 313-316]. This novel functional connexion of active sites must be brought about by transacetylation reactions between lipoic acid residues of neighbouring E2 chains in the enzyme complex. There was also a slow transacylation process between the rapidly acetylated lipoic acid residues and those that did not react in the initial, faster phase. This interaction was not investigated in detail, since it is too slow to be of kinetic significance in the normal enzymic reaction.     

Temperature-dependence of intramolecular coupling of active sites in pyruvate dehydrogenase multienzyme complexes.

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes of Escherichia coli, ox heart and Bacillus stearothermophilus was measured at various temperatures. As the temperature was raised, the extent of active-site coupling was found to increase, approaching a maximum near the physiological growth temperature of the organism. Under these conditions, a single pyruvate dehydrogenase (lipoamide) dimer appeared able to cause a rapid (20s) reductive acetylation of probably all 24 polypeptide chains in the dihydrolipoamide acetyltransferase core of the enzyme complex from E. coli at 37 degrees C, and of most if not all of the 60 polypeptide chains in the dihydrolipoamide acetyltransferase cores of the enzymes from ox heart and B. stearothermophilus at 37 degrees C and 60 degrees C respectively. Experiments designed to measure the inter-core and intra-core migration of enzyme subunits suggested that, in the bacterial enzymes at least, this was not a major contributor to active-site coupling.     

Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes.

The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.     

Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components

Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess ‘moonlighting’ activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions. Various known affectors of physiological activity and ROS production, including Ca²⁺, ADP, lipoylation status or pH, were investigated. The human complexes were also reconstituted with the most prevalent human pathological mutant of the E3 component, G194C and characterized; isolated human E3 with the G194C substitution was previously reported to have an enhanced ROS generating capacity. It is demonstrated that: i. PDHc, similarly to OGDHc, is able to generate ROS and this feature is displayed by both the E. coli and human complexes, ii. Reconstituted hPDHc generates ROS at a significantly higher rate as compared to hOGDHc in both the forward and the reverse reactions when ROS generation is calculated for unit mass of their common E3 component, iii. The E1 component or E1-E2 subcomplex generates significant amount of ROS only in hOGDHc; iv. Incorporation of the G194C variant of hE3, the result of a disease-causing mutation, into reconstituted hOGDHc and hPDHc indeed leads to a decreased activity of both complexes and higher ROS generation by only hOGDHc and only in its reverse reaction.     

Investigation of the mechanism of active site coupling in the pyruvate dehydrogenase multienzyme complex of Escherichia coli by protein engineering

Site-directed mutagenesis of the aceF gene of Escherichia coli was used to generate a nested set of deletions in the long (alanine + proline)-rich sequence that separates the lipoyl domain from the dihydrolipoamide dehydrogenase-binding domain in the "one-lipoyl domain" dihydrolipoamide acetyltransferase polypeptide chains of a pyruvate dehydrogenase multienzyme complex. The deletions reduced the number of residues in this sequence successively from 32 to 20, 13, 7 and just 1 residue. In all instances, pyruvate dehydrogenase complexes were still assembled in vivo around cores containing the deleted chains, and those with the two shortest deletions were essentially fully active. However, the two most severe deletions caused falls of 50% or more in specific catalytic activity. Similarly, although shortening the interdomain sequence to 20 residues left the system of active-site coupling unimpaired, cutting it to 13 residues or less caused substantial falls in the reductive acetylation of the lipoyl domains and corresponding losses of active-site coupling. The changes in specific catalytic activity and active-site coupling that accompanied the shortening of the (alanine + proline)-rich segment were reflected in the poorer growth rates of the relevant strains of E. coli on stringent substrates. All these results are consistent with this (alanine + proline)-rich sequence acting as a linker region that facilitates the movements of the lipoyl domains required for full catalytic activity and active-site coupling in the complex. The other two such sequences that separate the additional lipoyl domains in the N-terminal half of the wild-type "three-lipoyl domain" dihydrolipoamide acetyltransferase chain are presumed to function similarly. This role is consistent with the conformational flexibility assigned to these segments from previous studies based on 1H nuclear magnetic resonance spectroscopy and protein engineering.     

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli.

Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichia coli. In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively. Chimeric E2p components were constructed by exchanging the three domains between E2p from A. vinelandii and E. coli on gene level. The six chimeric E2p proteins were expressed and purified from E. coli TG2. All chimeras were catalytically active, 24-subunit E2p proteins. Interactions of the peripheral components E1p and E3 with the wild-type enzymes from A. vinelandii and E. coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex. A. vinelandii E3 interacts only with those chimeras that contain the A. vinelandii binding domain, whereas E. coli E3 interacts with all chimeras. Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3. Recognition of E1p depends on the origin of both the binding domain and the catalytic domain. E. coli E1p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E. coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E. coli E2p. No binding of E. coli E1p was observed when both domains were of A. vinelandii origin. A. vinelandii E1p recognizes E2p from A. vinelandii and E. coli, but strong interaction required that the binding and catalytic domain were of the same origin. Exchange of lipoyl domains had no effect on the binding properties of the E1p component. These observations confirm previous conclusions, based on site-directed mutagenesis of A. vinelandii E2p [Schulze, E., Westphal, A. H., Boumans, H., and de Kok, A. (1991) Eur. J. Biochem. 202, 841-848], that the binding site for E1p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E. coli E2p. Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A. vinelandii E2p. Dissociation depends on the binding of peripheral components to the E1p-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis.

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.     

Substrate-induced structural changes of the pyruvate dehydrogenase multienzyme complex

The time course of the overall reaction catalyzed by the pyruvate dehydrogenase multienzyme complex produces an unexpectedly high lag (tau = 8 S) even in the presence of saturating concentrations of its substrates. The preincubation of the pyruvate dehydrogenase complex with one of the substrates alone decreases the duration of this lag, and all the substrates of the pyruvate dehydrogenase component (E1) and dihydrolipoyl transacetylase component (E2) together (pyruvate, thiamine pyrophosphate, and CoA) result in the complete disappearance of the lag. The reduction of the dihydrolipoyl dehydrogenase component (E3) of the pyruvate dehydrogenase complex with the substrates of the complex in the absence of NAD+ produces significantly different quenching in the FAD fluorescence, and then the reduction with the substrates of E3 as dihydrolipoic acid and dithioerythritol. (The formation of FADH2 was not observed in the system.) The higher fluorescence quenching in the presence of substrates of pyruvate dehydrogenase complex compared to the effect caused by the substrates of the E3 component (dihydrolipoic acid and DTE) indicates conformational changes additionally manifested in the fluorescence properties of the enzyme complex. The substrate-induced quenching of the enzyme-bound FAD fluorescence shows biphasic kinetics. The rate constant of the slow phase is comparable with the rate constant calculated from the time duration of the lag phase observed in the overall reaction. The kinetic analysis of both intensity and anisotropy decrease of the FAD fluorescence suggests a consecutive transmittance of an all substrate-coordinated, induced conformational changes directed from the pyruvate dehydrogenase-via the lipoyl transacetylase--to the lipoyl dehydrogenase. Two simultaneous conformational effects caused by binding of the substrates can be distinguished; one of them results the fluorescence of the bound FAD to be more quenched, while the other makes the FAD more mobile. The first-order rate constants of both these conformational changes were determined. The present observations suggest that the pyruvate dehydrogenase complex exists in a partially inactive state in the absence of its substrates, and it becomes active due to conformational changes caused by the binding of its substrates.     

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The hydrogenase from Paracoccus denitrificans, which is an intrinsic membrane protein, has been solubilised from membranes by Triton X-100. The partial specific volume of the solubilised protein has been determined using sucrose density gradient centrifugation in H2O and 2H2O. The values of the specific volumes of hydrogenase, measured in the presence or absence of Triton X-100, are 0.73 and 0.74 ml . g-1, respectively, indicating that hydrogenase binds much less than one micelle of Triton X-100. The sedimentation coefficient of hydrogenase is increased from 10.4 S to 15.9 S on removal of detergent. The Stokes' radius of hydrogenase, determined by gel filtration on Sepharose 6B, is 5.5 nm in the presence of Triton X-100 compared to 6.7 nm in the absence of detergent. The apparent molecular weight therefore increases from 242,500 to 466,000 on removal of detergent. In the presence of urea and sodium dodecylsulphate, the hydrogenase has an apparent molecular weight of 63,000. The enzyme therefore behaves as a non-covalently linked tetramer in the presence of Triton X-100. Removal of Triton X-100 results in association of tetramers to form octamers.     

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Bacillus subtilis.

Limited digestion of the pyruvate dehydrogenase complex of Bacillus subtilis with either trypsin or chymotrypsin at 0 degrees C inhibited its ability to decarboxylate pyruvate and 2-oxoisovalerate oxidatively, without causing disassembly of the complex. The proteinases selectively cleaved the E1 alpha subunits to form two fragments of Mr 31500 and approx. 9500, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both fragments remaining bound to the complex. Trypsin also caused a much slower cleavage of the E2 subunits, to form a fragment of apparent Mr 34000. The inhibition of overall dehydrogenase-complex activity was accompanied by the apparent loss of the pyruvate-driven and 2-oxoisovalerate-driven E1 activities, which was found to be due to a large increase in the Km for the 2-oxo acids: this change was correlated with the cleavage of the E1 alpha subunit.     

Antibodies against an inter-domain segment of polypeptide chain inhibit active-site coupling in the pyruvate dehydrogenase multienzyme complex

A synthetic peptide, AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKA, proved to be an efficient and specific immunogen in rabbits. The amino acid sequence of the peptide is identical to that of the inter-domain region (PEP3) linking the innermost of the three lipoyl domains to the dihydrolipoamide dehydrogenase-binding domain in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli. Fab fragments from anti-PEP3 antibodies selectively inhibited active-site coupling in the complex without affecting the individual activities of the three component enzymes, highlighting the role of the inter-domain regions as flexible linkers in catalysis.     

Repeating functional domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Each polypeptide chain in the lipoate acetyltransferase (E2) core of the pyruvate dehydrogenase complex from Escherichia coli contains three repeating sequences in the N-terminal half of the molecule. The repeats are highly homologous in primary structure and each includes a lysine residue that is a potential site for lipoylation. We have shown that all three sites are lipoylated, at least in part, and that the three lipoylated segments of the E2 chain can be isolated as distinct functional domains after limited proteolysis. Each domain becomes partly acetylated in the intact complex in the presence of substrate. In the primary structure, the domains are separated by regions of polypeptide chain oddly rich in alanine and proline residues. These regions are probably the conformationally mobile segments observed in the 1H-n.m.r. spectrum of the complex and which are removed by tryptic cleavage at Lys-316. The C-terminal half of the molecule contains the acetyltransferase active site and the binding sites for E1, E3 and other E2 subunits. The pyruvate dehydrogenase complex of E. coli, which has a heterogeneous quaternary structure, is thus far unique among the 2-oxo acid dehydrogenase complexes in possessing more than one lipoyl domain per E2 chain, but this may be a general feature of the enzyme from Gram-negative organisms.     

Tellurite-induced carbonylation of the Escherichia coli pyruvate dehydrogenase multienzyme complex

The soluble tellurium oxyanion, tellurite, is toxic for most organisms. At least in part, tellurite toxicity involves the generation of oxygen-reactive species which induce an oxidative stress status that damages different macromolecules with DNA, lipids and proteins as oxidation targets. The objective of this work was to determine the effects of tellurite exposure upon the Escherichia coli pyruvate dehydrogenase (PDH) complex. The complex displays two distinct enzymatic activities: pyruvate dehydrogenase that oxidatively decarboxylates pyruvate to acetylCoA and tellurite reductase, which reduces tellurite (Te⁴⁺) to elemental tellurium (Te^o). PDH complex components (AceE, AceF and Lpd) become oxidized upon tellurite exposure as a consequence of increased carbonyl group formation. When the individual enzymatic activities from each component were analyzed, AceE and Lpd did not show significant changes after tellurite treatment. AceF activity (dihydrolipoil acetyltransferase) decreased ~30% when cells were exposed to the toxicant. Finally, pyruvate dehydrogenase activity decreased >80%, while no evident changes were observed in complex′s tellurite reductase activity.     

Molecular weight and symmetry of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The molecular weight and polypeptide chain stoichiometry of the native pyruvate dehydrogenase multienzyme complex from Escherichia coli were determined by independent techniques. The translational diffusion coefficient (D^o_20,w) of the complex was measured by laser light intensity fluctuation spectroscopy and found to be 0.90 (±0.02) × 10^?11m²/s. When this was combined in the Svedberg equation with the measured sedimentation coefficient (s^o_20,w = 60.2 (±0.4) S) and partial specific volume ($\text{v}\text{?}\text{= 0.735 (±0.01)}\text{ml/g}$), the molecular weight of the intact native complex was calculated to be 6.1 (±0.3) × 10⁶. The polypeptide chain stoichiometry (pyruvate decarboxylase: lipoate acetyltransferase: lipoamide dehydrogenase) of the same sample of pyruvate dehydrogenase complex was measured by the radioamidination technique of Bates et al. (1975) and found to be 1.56:1.0:0.78.From this stoichiometry and the published polypeptide chain molecular weights estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, a minimum chemical molecular weight of 283,000 was calculated. This structure must therefore be repeated approximately 22 times to make up the native complex, a number which is in good agreement with the expected repeat of 24 times if the lipoate acetyltransferase core component has octahedral symmetry. It is consistent with what appears in the electron microscope to be trimer-clustering of the lipoate acetyltransferase chains at the corners of a cube. It rules out any structure based on 16 lipoate acetyltransferase chains comprising the enzyme core.The preparation of pyruvate dehydrogenase complex was polydisperse: in addition to the major component, two minor components with sedimentation coefficients (s^o_20,w) of 90.3 (±0.9) S and 19.8 (±0.3) S were observed. Together they comprised about 17% of the total protein in the enzyme sample. Both were in slowly reversible equilibrium with the major 60.2 S component but appeared to be enzymically active in the whole complex reaction. The faster-sedimenting species is probably a dimer of the complex, whereas the slower-sedimenting species has the properties of an incomplete aggregate of the component enzymes of the complex based on a trimer of the lipoate acetyltransferase chain.