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PubMed1986

Immunochemical analysis of normal and mutant forms of human pyruvate dehydrogenase.

Pyruvate dehydrogenase (PDH) deficiency has been described in many patients with primary lactic acidosis. However, there are very few cases in which a structural defect in the complex has been clearly demonstrated. Measurement of the activity of the PDH complex in cultured human cells has proved unreliable, and a combination of structural and functional studies are required to make a definitive diagnosis. For this reason, an immunochemical strategy has been developed to complement direct enzyme assay in the detection and further characterization of PDH deficiency. We illustrate the usefulness of this approach by describing defects in the alpha-subunit of the pyruvate decarboxylase component of the PDH complex in two patients with primary lactic acidosis. In one patient, there is no immunologically cross-reacting material corresponding to this subunit. In the second patient, there appears to be an intrinsic structural defect in the subunit which restricts dephosphorylation (and hence activation) of the inactive phosphorylated complex.

Acidosis, Lactic↗

PubMed1980

Elementary steps in the reaction mechanism of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: kinetics of acetylation and deacetylation.

The kinetics of the acetylation of the pyruvate dehydrogenase complex from Escherichia coli by [3-14C]pyruvate and of the deacetylation of the complex by coenzyme A have been studied by using rapid mixing-quench techniques. The time course for acetylation in 4 mM thiamin pyrophosphate, 2 mM MgSO4, and 0.02 M potassium phosphate (pH 7.0) at 4 degrees C can be analyzed in terms of two kinetic processes. At long times 10 nmol of acetyl groups is incorporated per mg of enzyme complex (48 sites per complex of molecular weight 4.8 X 10(6)). The slower process is much too slow to be of catalytic significance. The rate constant for the faster process is not dependent on enzyme concentration and reaches a limiting value of 40--65 s-1 at high pyruvate concentrations; the exact value is dependent on the detailed acetylation mechanism assumed. The minimum molar turnover number of the enzyme complex is 420 s-1 (17.5 s-1 per pyruvate decarboxylase). The acetylated lipoic acids are deacetylated by coenzyme A at a rate much faster than that of acetylation. Complete deacetylation is obtained only if the deacetylation is carried out within seconds of the acetylation, apparently because dead-end intramolecular transfers of acetyl groups from the lipoic acids to other functional groups on the enzyme not essential for catalytic activity can occur. The results obtained suggest only about half of the acetylation reactions are on the main catalytic pathway.

Acetylation↗

PubMed2002

Enzymatic (R)-phenylacetylcarbinol production in benzaldehyde emulsions.

(R)-Phenylacetylcarbinol [(R)-PAC)] is the chiral precursor for the production of the pharmaceuticals ephedrine and pseudoephedrine. Reaction conditions were improved to achieve increased (R)-PAC levels in a simple batch biotransformation of benzaldehyde emulsions and pyruvate, using partially purified pyruvate decarboxylase (PDC) from the filamentous fungus Rhizopus javanicus NRRL 13161 as the catalyst. Lowering the temperature from 23 degrees C to 6 degrees C decreased initial rates but increased final (R)-PAC concentrations. Addition of ethanol, which increases benzaldehyde solubility, was not beneficial for (R)-PAC production. It was established that proton uptake during biotransformation increases the pH above 7 thereby limiting (R)-PAC production. For small-scale studies, biotransformations were buffered with 2-2.5 M MOPS (initial pH 6.5). High concentrations of MOPS as well as some alcohols and KCl stabilised PDC. A balance between PDC and substrate concentrations was determined with regards to ( R)-PAC production and yields on enzyme and substrates. R. javanicus PDC (7.4 U/ml) produced 50.6 g/l (337 mM) ( R)-PAC in 29 h at 6 degrees C with initial 400 mM benzaldehyde and 600 mM pyruvate. Molar yields on consumed benzaldehyde and pyruvate were 97% and 59%, respectively, with 17% pyruvate degraded and 24% converted into acetaldehyde and acetoin; 43% PDC activity remained, indicating reasonable enzyme stability at high substrate and product concentrations.

Acetone↗

PubMed1994

The refined structures of a stabilized mutant and of wild-type pyruvate oxidase from Lactobacillus plantarum.

The crystal structure of pyruvate oxidase (EC 1.2.3.3) from Lactobacillus plantarum stabilized by three point mutations has been refined at 2.1 A resolution using the simulated annealing method. Based on 87,775 independent reflections in the resolution range 10 to 2.1 A, a final R-factor of 16.2% was obtained at good model geometry. The wild-type enzyme crystallizes isomorphously with the stabilized enzyme and has been analyzed at 2.5 A resolution. Pyruvate oxidase is a homotetramer with point group symmetry D2. One 2-fold axis is crystallographic, the others are local. The crystallographic asymmetric unit contains two subunits, and the model consists of the two polypeptide chains (residues 9 through 593), two FAD, two ThDP*Mg2+ and 739 water molecules. Each subunit has three domains; the CORE domain, the FAD domain and the ThDP domain. The FAD-binding chain fold is different from those of other known flavoproteins, whereas the ThDP-binding chain fold resembles the corresponding folds of the two other ThDP enzymes whose structure is known, transketolase and pyruvate decarboxylase. The peptide environment most likely forces the pyrimidine ring of ThDP into an unusual tautomeric form, which is required for catalysis. The structural differences between the wild-type and the stabilized enzyme are small. All three point mutations are at or near to the subunit interfaces, indicating that they stabilize the quarternary structure as had been deduced from reconstitution experiments.

Amino Acid Sequence↗

PubMed1980

Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides.

1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.

Amino Acids↗

PubMed1973

Biosynthesis of branched-chain amino acids in Schizosaccharomyces pombe: properties of acetohydroxy acid synthetase.

The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.

Acetates↗

PubMed1982

Quaternary structure of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus studied by a new reversible cross-linking procedure with bis(imidoesters).

The bis(amidine) cross-links formed between protein subunits by treating them with bis(imidoesters) were found to be rapidly broken by exposing the cross-linked proteins to methylamine buffers containing the aprotic solvent acetonitrile. This cleavage step could be introduced between the two dimensions of a diagonal gel electrophoretic separation of cross-linked proteins to facilitate identification of the contributors to a cross-linked species. Tests with the tetrameric enzyme glyceraldehyde-3-phosphate dehydrogenase demonstrated the simplicity and effectiveness of the technique. When the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was treated with a range of bis(imidoesters), from dimethyl succinimidate to dimethyl suberimidate, the most informative set of products was obtained with dimethyl glutarimidate. The longer bis(imidoesters) caused too extensive cross-linking of the enzyme subunits, although the beta chain of the pyruvate decarboxylase component always appeared to be the most resistant. Almost all the cross-linked species up to pentamers of the lipoate acetyltransferase polypeptide chain (apparent Mr approximately 280 000) were identified by means of the diagonal gel electrophoretic procedure after cleavage of the cross-links. The introduction of the methylamine cleavage step enables the bis(imidoester) for such experiments to be selected purely for the efficacity of its cross-linking reaction with the protein and dispenses with the need to incorporate a specially cleavable bond in the reagent.

Chemical Phenomena↗

PubMed1979

Subunit stoichiometry and molecular weight of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

The molar ratio of the component enzymes of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was found to be 1.8:1.7:1[pyruvate decarboxylase (E1):dihydrolipoyl transacetylase (E2):dihydrolipoyl dehydrogenase (E3)]. This ratio was determined by measuring the Coomassie blue staining of the constituent enzymes after sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. The above ratio is the average of four separate experiments with two different enzyme preparations. The average molecular weights of the individual enzymes were found to be 96,000, 76,000, and 55,000 for E1, E2, and E3, respectively, by sodium dodecyl sulfate and sodium dodecyl sulfate/8 M urea polyacrylamide gel electrophoresis and by column chromatography in 6 M guanidine . HCl. The molecular weight of E2 was reduced to 33,000-36,000 after extensive reduction and alkylation with iodoacetamide. The molecular weights of the complex, E1, and E3 were found to be 4,800,000, 182,000, and 104,000, respectively, with low-angle laser light scattering. Both E1 and E3 are dimeric under the conditions employed. If octahedral symmetry is assumed for the E2 core, a polypeptide chain ratio of 24:24:12 (E1:E2:E3) is in good agreement with the measured molar ratio of component enzymes and the molecular weight of the pyruvate dehydrogenase complex.

Coloring Agents↗

PubMed1998

Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation.

The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.

Aerobiosis↗

PubMed2006

Distinct modes of recognition of the lipoyl domain as substrate by the E1 and E3 components of the pyruvate dehydrogenase multienzyme complex.

Two-dimensional (15)N-heteronuclear single-quantum coherence (HSQC) NMR studies with a di-domain (lipoyl domain+ linker+ peripheral subunit-binding domain) of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus allowed a molecular comparison of the need for lipoic acid to be covalently attached to the lipoyl domain in order to undergo reductive acetylation by the pyruvate decarboxylase (E1) component, in contrast with the ability of free lipoic acid to serve as substrate for the dihydrolipoyl dehydrogenase (E3) component. Tethering the lipoyl domain to the peripheral subunit-binding domain in a complex with E1 or E3 rendered the system more like the native enzyme complex, compared with the use of a free lipoyl domain, yet of a size still amenable to investigation by NMR spectroscopy. Recognition of the tethered lipoyl domain by E1 was found to be ensured by intensive interaction with the lipoyl-lysine-containing beta-turn and with residues in the protruding loop close to the beta-turn. The size and sequence of this loop varies significantly between species and dictates the lipoylated lipoyl domain as the true substrate for E1. In contrast, with E3 the main interaction sites on the tethered lipoyl domain were revealed as residues Asp41 and Ala43, which form a conserved sequence motif, DKA, around the lipoyl-lysine residue. No domain specificity is observed at this step and substrate channelling in the complex thus rests on the recognition of the lipoyl domain by the first enzyme, E1. The cofactor, thiamine diphosphate, and substrate, pyruvate, had distinct but contrasting effects on the E1/di-domain interaction, whereas NAD(+) and NADH had negligible effect on the E3/di-domain interaction. Tethering the lipoyl domain did not significantly change the nature of its interaction with E1 compared with a free lipoyl domain, indicative of the conformational freedom allowed by the linker in the movement of the lipoyl domain between active sites.

Amino Acid Motifs↗

PubMed1978

Fluorescence energy-transfer studies on the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii.

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (greater than or equal to 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis. Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.

Acetylcysteine↗

PubMed2005

The molecular origins of specificity in the assembly of a multienzyme complex.

The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a "charge zipper" of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the approximately 9 MDa PDH complex.

Binding Sites↗

PubMed1989

Neonatal pyruvate dehydrogenase deficiency with lipoate responsive lactic acidaemia and hyperammonaemia.

A 2-day-old girl developed a severe lactic acidosis with a normal lactate/pyruvate ratio and hyperammonaemia. Plasma arginine and citrulline levels were below the limit of detection. In muscle total pyruvate dehydrogenase complex (PDHC) and pyruvate decarboxylase (E1) activities were reduced to a fraction of lower control values. The acute neonatal period was bridged with peritoneal dialysis, dichloroacetate therapy, supplements of arginine and branched chain amino acids, a complete vitamin B complex and lipoic acid. Lactate homeostasis responded to pharmacological supplements of lipoic acid. At age 1 year the child was hypotonic, showed severe developmental retardation, optic atrophy and cranial dysmorphism. She died aged 1 year 8 months with signs of respiratory paralysis but with normal lactate levels under assisted breathing. Pathological findings at autopsy were suggestive of Leigh syndrome, interstitial pneumonia and extensive fatty infiltration of hepatocytes. Regression analysis of data from 187 plasma amino acid determinations from the patient over a period of 1 year 8 months revealed a persistent-imbalance involving alanine, glutamic acid, glutamine, proline, citrulline and branched chain amino acids. Aspects of acute and long-term therapy in this patient and some implications of the imbalances in plasma amino acids are discussed.

Acidosis, Lactic↗

PubMed2006

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.

Animals↗

PubMed2003

Epitope mapping on E1alpha subunit of pyruvate dehydrogenase complex with autoantibodies of patients with primary biliary cirrhosis.

BACKGROUND: A major mitochondrial autoantigen recognized by sera of patients with primary biliary cirrhosis (PBC) is dihydrolipoamide acetyltransferase (E2) of the pyruvate dehydrogenase complex (PDH). The alpha subunit of pyruvate decarboxylase (E1alpha) of PDH is also recognized in some E2-reactive PBC sera, suggesting that the occurrence of autoimmunity against Elalpha is subsequent to that against E2. METHODS. To investigate the mechanism inducing autoimmunity against E1alpha, we surveyed immunoreactive sequences of E1alpha by ELISA with synthesized oligopeptides, and determined minimum amino acid residues for each determinant. RESULTS: The major determinants of E1alpha appeared to reside in its N-terminal region, apparently forming 'nested epitopes', and all E1alpha-reactive PBC sera tested recognized these regions. Minor epitopes were also found scattered throughout the entire sequence. The reactivities of these minor epitopes to individual PBC sera were proportional to those of the major epitopes. All the epitopes were located in hydrophilic regions of E1alpha, and many of them were out of the known functional domains (TPP-binding domain, subunit interaction site, and phosphorylation sites) whose structures are phylogenically well conserved. Furthermore, the sequences of many epitopes appeared to be specific to humans. CONCLUSION: These observations suggest that determinant spreading might underlie the autoimmunity against Elalpha.

Amino Acid Sequence↗

PubMed1968

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria.

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate-cytochrome c oxidoreductase and NADH-cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP(+)-linked) and NADPH-cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD(+)- and NADP(+)-linked), alpha-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD(+)-linked), glutamate-oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, alpha-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD(+)- and NADP(+)-linked), glutamate dehydrogenase (NAD(+)-linked), glutamate-oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.

Alcohol Oxidoreductases↗

PubMed1971

Metabolism of acetoin in mammalian liver slices and extracts. Interconversion with butane-2,3-diol and biacetyl.

1. [(14)C]Acetoin was enzymically synthesized from [(14)C]pyruvate with a pyruvate decarboxylase preparation. Its optical activity was [alpha](20) (d)-78 degrees . 2. Large amounts (1000-fold higher than physiological concentrations) of acetoin were incubated with rat liver mince. Acetoin disappeared but very little (14)CO(2) was evolved. A compound accumulated, which was purified and identified as butane-2,3-diol. Chromatography on borate-impregnated paper indicated the presence of both the erythro and threo forms. 3. Liver extracts capable of interconverting biacetyl, acetoin and butane-2,3-diol were obtained. These interconversions were catalysed by two different enzymes: acetoin dehydrogenase (EC 1.1.1.5) and butane-2,3-diol dehydrogenase (EC 1.1.1.4), previously identified in bacteria. Both required NAD(+) or NADP(+) as cofactors and were different from alcohol dehydrogenase. The equilibrium in both cases favoured the more reduced compound. 4. The activity of butane-2,3-diol dehydrogenase was decreased by dialysis against EDTA: the addition of Co(2+), Cu(2+), Zn(2+) and other bivalent metal ions restored activity. 5. Biacetyl reductase was resolved into multiple forms by CM-Sephadex chromatography and electrophoresis.

Alcohol Oxidoreductases↗

PubMed1996

Protein-protein interactions in the pyruvate dehydrogenase multienzyme complex: dihydrolipoamide dehydrogenase complexed with the binding domain of dihydrolipoamide acetyltransferase.

BACKGROUND: The ubiquitous pyruvate dehydrogenase multienzyme complex is built around an octahedral or icosahedral core of dihydrolipoamide acetyltransferase (E2) chains, to which multiple copies of pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) bind tightly but non-covalently. E2 is a flexible multidomain protein that mediates interactions with E1 and E3 through a remarkably small binding domain (E2BD). RESULTS: In the Bacillus stearothermophilus complex, the E2 core is an icosahedral assembly of 60 E2 chains. The crystal structure of the E3 dimer (101 kDa) complexed with E2BD (4 kDa) has been solved to 2.6 A resolution. Interactions between E3 and E2BD are dominated by an electrostatic zipper formed by Arg135 and Arg139 in the N-terminal helix of E2BD and Asp344 and Glu431 of one of the monomers of E3. E2BD interacts with both E3 monomers, but the binding site is located close to the twofold axis. Thus, in agreement with earlier biochemical results, it is impossible for two molecules of E2BD to bind simultaneously to one E3 dimer. CONCLUSIONS: Combining this new structure for the E3-E2BD complex with previously determined structures of the E2 catalytic domain and the E2 lipoyl domain creates a model of the E2 core showing how the lipoyl domain can move between the active sites of E2 and E3 in the multienzyme complex.

Acetyltransferases↗