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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↗

PubMed2003

Prediction of the binding site on E1 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The beta-subunit (E1beta) of the pyruvate decarboxylase (E1, alpha(2)beta(2)) component of the Bacillus stearothermophilus pyruvate dehydrogenase complex was comparatively modelled based on the crystal structures of the homologous 2-oxoisovalerate decarboxylase of Pseudomonas putida and Homo sapiens. Based on this homology modelling, alanine-scanning mutagenesis studies revealed that the negatively charged side chain of Glu285 and the hydrophobic side chain of Phe324 are of particular importance in the interaction with the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the complex. These results help to identify the site of interaction on the E1beta subunit and are consistent with thermodynamic evidence of a mixture of electrostatic and hydrophobic interactions being involved.

Alanine↗

PubMed1984

A coupled fluorometric rate assay for pyruvate dehydrogenase in cultured human fibroblasts.

A method for measuring the activity of the pyruvate dehydrogenase complex (PDC) by coupling acetyl-CoA production to acetylation of a fluorescent dye is described. Acetylation of cresyl violet acetate by pigeon liver acetyltransferase results in a shift of its fluorescence spectrum from lambda ex max = 575, lambda em max = 620 nm to lambda ex max = 475, lambda em max = 575 nm. The rate of appearance of acetylated dye was followed fluorometrically and was proportional to PDC activity in extracts of cultured human fibroblasts. The assay showed appropriate substrate and cofactor dependence and had a working range between 0.04 and 70 munits. It is 10 times more sensitive than the spectrophotometric assay on which it is based (working range 0.4-31 munits) and is equally convenient. Unactivated PDC activity in fibroblast extracts was 0.75 (0.60-0.92) munits/mg protein (mean and range for six cell lines).

Acetyl Coenzyme A↗

PubMed2006

Ligand-induced effects on pyruvate dehydrogenase kinase isoform 2.

Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/L0.5 estimates: approximately 53%/3 microM for ATP; approximately 49%/15 microM for ADP; and approximately 71%/approximately 590 microM for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and > or = 80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 microM pyruvate, > or = 150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2(red)) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2(red) was modestly hindered by 200 microM level of ATP or ADP or 5.0 mM pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 microM levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2(red) may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.

Adenosine Diphosphate↗

PubMed1995

Dissociation and unfolding of the pyruvate dehydrogenase complex by guanidinium chloride.

The effect of guanidinium chloride (GdnHCl) on the pyruvate dehydrogenase complex (PDC) from bovine heart and its constituent enzymes has been studied. The overall activity of the complex is lost reversibly at low levels of GdnHCl (0.2 M) which cause 40-50% inactivation but no loss of overall secondary or tertiary structures of the individual enzymes; the inactivation of the complex is shown to be caused by dissociation of the E1 and E3 components from the E2/X core assembly. This provides an improved procedure for controlled dissociation of the complex and efficient recovery of its component enzymes in their native states. Higher concentrations of GdnHCl (up to 4 M) lead to the unfolding and irreversible inactivation of the separate enzymes of the complex with the E2/X core proving the most resistant to GdnHCl-induced unfolding. Neither the 60-meric E2/X core assembly nor the dimeric E3 component are dissociated into monomers in the presence of 6 M GdnHCl; the latter enzyme forms higher-M(r) aggregates under these conditions.

Animals↗

PubMed1992

Molecular genetic aspects of human pyruvate dehydrogenase and its defect.

Genomic clones encompassing the entire genes for the human pyruvate dehydrogenase alpha and beta subunits (PDH alpha or beta) have been isolated by screening the leukocyte genomic libraries with a nick-translated human foreskin fibroblast PDH alpha or beta cDNA probe. These genomic clones were characterized by restriction enzyme analysis. extensive DNA sequencing and primer extension analysis. The PDH alpha gene spans 17.08 kilobases and is composed of 11 exons and 10 introns within its coding region. The 18-kilobase clone of PDH beta gene is composed of 10 exons and 9 introns. All intron-exon splice junctions of two genes follow the GT/AG rule. A total of seven Alu repeats in the PDH alpha gene were found in five introns and two Alu family in the PDH beta gene were found in intron 2 and 8. The 5'-flanking region of the PDH alpha gene contains typical CCAAT and TATA-like consensus promoter sequence and two Sp1 binding sequences. That of the PDH beta gene contains a TCAAT sequence but no TATA sequence. Primer extension analyses indicated that the PDH alpha and beta genes transcription start sites are thymine and adenine residues located 124 and 132 bases upstream from initiation codon in exon 1, respectively. Genomic DNA of patient, died 93 hours after birth with acidemia and defect of PDH activity, was isolated and all of the exons of PDH alpha and beta genes were amplified by PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1985

Insulin mediator stimulates pyruvate dehydrogenase of intact liver mitochondria.

The effects of putative insulin mediators on the pyruvate dehydrogenase (PDH) activity of intact mitochondria isolated from rat liver were investigated. The mitochondria were judged intact on the basis of electron microscopic examination and demonstrated respiratory control. Only mitochondria having respiratory control ratios of greater than 4, using succinate as a substrate, were used in these studies. Addition of physiologic concentrations of insulin to these mitochondria caused stimulation of PDH activity, attributed to generation of an insulin mediator from plasma membranes contaminating the mitochondrial preparation. Exogenous plasma membranes from rat adipocytes or liver caused further stimulation of PDH activity, which was proportional to the amount of plasma membranes added. Addition of insulin to the mixture of mitochondria and plasma membranes stimulated PDH still further. The stimulation was proportional to the insulin concentration, with maximal effects observed at 50 microU/ml insulin. Partially purified mediators from liver, muscle, H4-II-E hepatoma cells, and IM9 lymphocytes also stimulated PDH activity in intact mitochondria. Mediators prepared from insulin-treated liver, muscle, and cultured hepatoma cells stimulated PDH more than did mediators from the corresponding untreated source. Mediator from insulin-treated IM9 lymphocytes stimulated PDH less than did mediator from untreated IM9 lymphocytes. These findings are consistent with the known effects of insulin on these tissues and with the reported effects of the various mediators on PDH activity in non-intact mitochondria. These observations support the proposal that these mediators are physiologically significant modulators of insulin's effects on PDH activity.

Animals↗

PubMed1981

Analysis of site occupancies in [32P]phosphorylated pyruvate dehydrogenase complexes by aspartyl-prolyl cleavage of tryptic phosphopeptides.

Activity of mammalian pyruvate dehydrogenase complexes from all sources so far tested is regulated by phosphorylation involving three sites. To facilitate understanding of the precise biological roles of the individual phosphorylation sites a method has now been developed, using pig heart [32P]phosphorylated complexes, which enables unambiguous measurement of their occupancies. Established methods of tryptic digestion give two peptides that contain the three phosphorylation sites: TA contains sites 1 and 2; TB contains site 3. Thus, while occupancy of site 3 may be determined unequivocally by tryptic digestion, occupancies of sites 1 and 2 cannot. The present paper shows that peptide TA may be specifically and quantitatively cleaved by formic acid at an Asp-Pro bond located between the two phosphorylation sites. Equal amounts of two new peptides each containing a different phosphorylation site (site 1 or site 2) are produced. The 32P-labelled peptides may be completely separated and quantified by high-voltage paper electrophoresis at pH 2. A combination of tryptic digestion (determination of 32P in site 3) and formic acid cleavage of peptide TA (determination of 32P in sites 1 and 2) thus enables unequivocal assignment of occupancies of individual phosphorylation sites to greater than 95% accuracy. This method has been used to show that during phosphorylation and inactivation of pig heart complexes (inactivated to between 1.5 % and 90%) greater than 98% of the observed inactivation was primarily attributable to phosphorylation of site 1; the contribution of site 2 was less than 2% if at all. Relative initial rates of phosphorylation site 1 X site 2 X site 3 were approximately 90:3:1.

Animals↗

PubMed1993

In vivo insulin sensitivity of the pyruvate dehydrogenase complex in tissues of the rat.

Activity of the insulin-activated pyruvate dehydrogenase complex (PDHC) is necessary for the complete oxidation of glucose to carbon dioxide or the conversion of glucose to fatty acids in lipogenic tissues. To determine the in vivo insulin sensitivity of PDHC activity in rat tissues, we measured the amount of PDHC in the active form in heart, diaphragm, red quadriceps, white adipose tissue (WAT), and brown adipose tissue (BAT) of rats exposed to five different circulating insulin concentrations under euglycemic clamp conditions. PDHC was measured in mitochondrial extracts of tissues rapidly dissected from rats in the starved state or after euglycemic clamp (4 mM) at insulin infusion rates of 0, 0.125, 0.25, and 2.0 U.kg-1.h-1. Increasing the insulin concentration increased the PDHC activity in all tissues, but the magnitude of this activation was different in different tissues (heart: 3.5-fold; diaphragm: 2.5-fold; red quadriceps: 1.8-fold; WAT: 3.4-fold; and BAT: 10.5-fold). Calculation of the half-maximal effective dose (ED50) for the activation of PDHC produced values that were similar in all tissues (heart: 112 pM; diaphragm: 108 pM; red quadriceps: 146 pM; WAT: 120 pM; and BAT: 118 pM). The insulin sensitivity of PDHC in these tissues correlated particularly well with the ED50 for the insulin effect of decreasing circulating nonesterified fatty acids (NEFA; 122 pM). The differences in the magnitude of the effect of increasing insulin on PDHC activity implies a tissue difference in the requirement for an increased capacity for glucose oxidation after insulin stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1981

The role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two lipoic acid residues on each dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were found to undergo oxidoreduction reactions with NAD+ catalysed by the lipoamide dehydrogenase component. It was observed that: (a) 2 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of acetyl-SCoA and NADH; (b) 4 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of NADH; (c) between 1 and 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with acetyl-SCoA plus NADH; (d) 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with pyruvate either before or after many catalytic turnovers through the overall reaction. There was no evidence to support the view that only half of the dihydrolipoic acid residues can be reoxidized by NAD+. However, chemical modification of lipoic acid residues with N-ethylmaleimide was shown to proceed faster than the accompanying loss of enzymic activity under all conditions tested, which indicates that not all the lipoyl groups are essential for activity. The most likely explanation for this result is an enzymic mechanism in which one lipoic acid residue can take over the function of another.

Acetyl Coenzyme A↗

PubMed1994

Postischemic inhibition of cerebral cortex pyruvate dehydrogenase.

Postischemic, mitochondrial respiratory impairment can contribute to prolonged intracellular lactic acidosis, secondary tissue deenergization, and neuronal cell death. Specifically, reperfusion-dependent inhibition of pyruvate dehydrogenase (PDH) may determine the degree to which glucose is metabolized aerobically vs. anaerobically. In this study, the maximal activities of pyruvate and lactate dehydrogenase (LDH) from homogenates of canine frontal cortex were measured following 10 min of cardiac arrest and systemic reperfusion from 30 min to 24 h. Although no change in PDH activity occurred following ischemia alone, a 72% reduction in activity was observed following only 30 min of reperfusion and a 65% inhibition persisted following 24 h of reperfusion. In contrast, no significant alteration in LDH activity was observed in any experimental group relative to nonarrested control animals. A trend toward reversal of PDH inhibition was observed in tissue from animals treated following ischemia with acetyl-L-carnitine, a drug previously reported to inhibit brain protein oxidation, and lower postischemic cortical lactate levels and improve neurological outcome. In vitro experiments indicate that PDH is more sensitive than LDH to enzyme inactivation by oxygen dependent free radical-mediated protein oxidation. This form of inhibition is potentiated by either elevated Ca2+ concentrations or substrate/cofactor depletion. These results suggest that site-specific protein oxidation may be involved in reperfusion-dependent inhibition of brain PDH activity.

Analysis of Variance↗

PubMed2005

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.

Binding Sites↗

PubMed2002

Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae.

The interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn-binding proteins (FnBPs) were purified to near homogeneity using Fn-coupled Sepharose 4B-affinity column chromatography, and amino acid sequence analysis of the 45 and the 30 kDa proteins identified them as elongation factor Tu (EF-Tu) and pyruvate dehydrogenase E1 beta subunit (PDH-B) respectively. The genes for EF-Tu and PDH-B were cloned, and the entire EF-Tu gene and NH2-terminus of PDH-B (NPDH (pyruvate dehydrogenase E1 beta subunit from amino acid 1-244)-B) gene were overexpressed in Escherichia coli. The recombinant proteins, rEF-Tu and rNPDH-B, were purified to homogeneity by His-tag affinity column chromatography and used to immunize rabbits. Purified rEF-Tu and rNPDH-B bound to Fn using a ligand immunoblot assay and ELISA. Immunogold electron microscopy with polyclonal antibodies reactive against rEF-Tu (antirEF-Tu) and rNPDH-B (antirNPDH-B) and whole cell radioimmunoprecipitation (WCRIP) revealed the surface location of these proteins. Adherence of viable M. pneumoniae to immobilized Fn was inhibited by antirEF-Tu and antirNPDH-B antisera in a dose-dependent and cumulative manner. These results demonstrate that M. pneumoniae EF-Tu and PDH-B, in addition to their major cytoplasmic biosynthetic and metabolic roles, can be surface translocated, which confers additional important biological functions.

Amino Acid Sequence↗

PubMed1997

Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae.

Stoichiometric analysis of pathways involved in anaerobic bioconversion of glycerol by Klebsiella pneumoniae revealed that enzyme(s) in addition to pyruvate formate-lyase (PFL) must be involved in pyruvate decarboxylation. In this work, enzymatic evidence is presented that confirmed a simultaneous involvement of pyruvate dehydrogenase complex (PDH) and excluded the presence of pyruvate:ferredoxin oxidoreductase in this anaerobic bioprocess. The in vitro PDH activity of cell extract from continuous culture was found to be strongly affected by the substrate (glycerol) concentration in medium and cell growth rate (dilution rate). It increases with increasing glycerol concentration and correlates well with the specific substrate uptake rate at different dilution rates in a kind of saturation function. At a similar substrate uptake rate, it decreases with cell growth rate. The in vitro activity of PDH is much higher than its in vivo activity calculated from the pathway stoichiometry but comparable to the calculated in vivo activity of PFL.

Anaerobiosis↗

PubMedDate not supplied

Pyruvate dehydrogenase complex: metabolic link to ischemic brain injury and target of oxidative stress.

The mammalian pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme complex (greater than 7 million Daltons) that catalyzes the oxidative decarboxylation of pyruvate to form acetyl CoA, nicotinamide adenine dinucleotide (the reduced form, NADH), and CO(2). This reaction constitutes the bridge between anaerobic and aerobic cerebral energy metabolism. PDHC enzyme activity and immunoreactivity are lost in selectively vulnerable neurons after cerebral ischemia and reperfusion. Evidence from experiments carried out in vitro suggests that reperfusion-dependent loss of activity is caused by oxidative protein modifications. Impaired enzyme activity may explain the reduced cerebral glucose and oxygen consumption that occurs after cerebral ischemia. This hypothesis is supported by the hyperoxidation of mitochondrial electron transport chain components and NAD(H) that occurs during reperfusion, indicating that NADH production, rather than utilization, is rate limiting. Additional support comes from the findings that immediate postischemic administration of acetyl-L-carnitine both reduces brain lactate/pyruvate ratios and improves neurologic outcome after cardiac arrest in animals. As acetyl-L-carnitine is converted to acetyl CoA, the product of the PDHC reaction, it follows that impaired production of NADH is due to reduced activity of either PDHC or one or more steps in glycolysis. Impaired cerebral energy metabolism and PDHC activity are associated also with neurodegenerative disorders including Alzheimer's disease and Wernicke-Korsakoff syndrome, suggesting that this enzyme is an important link in the pathophysiology of both acute brain injury and chronic neurodegeneration.

Animals↗

PubMed2005

Pyruvate fermentation by Oenococcus oeni and Leuconostoc mesenteroides and role of pyruvate dehydrogenase in anaerobic fermentation.

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate --> 1 lactate + 1 acetate + 1 CO(2)). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.

Anaerobiosis↗

PubMed1993

Partial activation of the pyruvate dehydrogenase kinase by the lipoyl domain region of E2 and interchange of the kinase between lipoyl domain regions.

The binding of the pyruvate dehydrogenase (E1) component and the E1-specific kinase to the core-forming dihydrolipoyl acetyltransferase (E2) component facilitates a severalfold enhancement in the rate at which the kinase phosphorylate E1 (i.e. versus free kinase phosphorylating free E1). The kinase and E1 associate with small exterior linker region-connected domains in the E2 structure. The kinase binds to one of two lipoyl domains, and the E1 component binds to a domain in E2"s structure between the lipoyl domain region and the inner domain. Sixty of the latter domains assemble to form a dodecahedron-shaped inner core. Binding of the kinase to a detached lipoyl domain region enhanced kinase activity. This bi-lipoyl domain fragment induced a 2-fold enhancement in the slow rate of phosphorylation of peptide substrate and intact E260 gave only a 50% higher rate. In contrast, the lipoyl domain fragment gave only a 40% enhancement in the faster rate of phosphorylation of E1; whereas the rate of phosphorylation of E1 was markedly increased (4-10-fold depending on conditions) by kinase interaction with the intact E2 core. Binding of E1 to an E2 structure lacking only the bi-lipoyl domain region did not enhance kinase activity. Thus, binding of the kinase to the lipoyl domain region elicits a structural change which enhances kinase activity; however, other processes are required to explain the very large enhancement in phosphorylation of E1 effected by intact E2 core. Among the latter is a need for a mechanism allowing one kinase molecule to phosphorylate many E1 tetramers, whereas both E1 and the kinase stay bound to the oligomeric E2 core (i.e. phosphorylation appears to be much faster than the dissociation of either the kinase or E1 tetramers from E260 core). Exposure of kinase bound to the lipoyl domain fragment to intact E2 core for 10 s allowed a transition to a maximal (7-fold) activation of the kinase. In the opposite direction, an increasing level of the free bi-lipoyl domain fragment rapidly reduced, in a concentration-dependent manner the activity of kinase bound initially to intact E2. The data strongly support kinase transfer between free lipoyl domains and the intact E2 core and fit about a 12-fold tighter binding of the kinase to intact E2 cores over binding to free lipoyl domains. Such an interchange of the kinase between these E2 structures was confirmed by sucrose gradient studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗

PubMed2002

Histidine 407, a phantom residue in the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex, activates reductive acetylation of lipoamide on the E2 subunit. An explanation for conservation of active sites between the E1 subunit and transketolase.

Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the reductive acetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit. While the H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca. 14% activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15% compared to parental E1. Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same time, mass spectrometric measurements clearly demonstrate much impaired reductive acetylation of the independently expressed lipoyl domain and of the intact 1-lipoyl E2 by the H407A variant compared to the parental E1. A proposal is presented to explain the remarkable conservation of the three-dimensional structure at the active centers of the E. coli E1 subunit and transketolase on the basis of the parallels in the ligation-type reactions carried out and the need to protonate a very weak acid, a dithiolane sulfur atom in the former, and a carbonyl oxygen atom in the latter.

Acetylation↗