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PubMed1983

Heterogeneity of binding sites for the pyruvate dehydrogenase component on the dihydrolipoyl transacetylase core of bovine kidney pyruvate dehydrogenase complex.

We have characterized the dissociation equilibrium constant (Kd) and the rate constants of association and dissociation for the binding of the pyruvate dehydrogenase component (PDH) to the dihydrolipoyl transacetylase component of kidney pyruvate dehydrogenase complex. We have found about 7 high-affinity sites (Kd = 1.5 X 10(-11) M) and about 13 low-affinity sites (Kd = 2.5 X 10(-8) M) with negative cooperativity for binding of PDH at the weaker sites. The high-affinity sites show a much higher rate constant for association of PDH with the core than do the low-affinity sites. Catalytic turnover strengthens binding of PDH at high-affinity sites when the complex is preincubated in the presence of thiamin pyrophosphate (TPP). In the absence of TPP, appreciably tighter binding occurs at the low-affinity sites (Kd = 5.9 X 10(-10) M). TPP weakens PDH binding at these sites with a half-maximal effect at about 5 microM TPP. In addition to TPP, increased ionic strength or 1-3 mM Mg2+ (with an enhanced effect of Mg2+ at higher ionic strengths) weakens PDH binding at low-affinity sites with a corresponding increase in the rate constant for dissociation. Further studies will be required to determine whether site heterogeneity contributes to dynamic processes in the function and regulation of the pyruvate dehydrogenase complex.

Acetyltransferases↗
PubMed2002

Pyruvate site of pyruvate phosphate dikinase: crystal structure of the enzyme-phosphonopyruvate complex, and mutant analysis.

Crystals of pyruvate phosphate dikinase in complex with a substrate analogue inhibitor, phosphonopyruvate (K(i) = 3 microM), have been obtained in the presence of Mg(2+). The structure has been determined and refined at 2.2 A resolution, revealing that the Mg(2+)-bound phosphonopyruvate binds in the alpha/beta-barrel's central channel, at the C-termini of the beta-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but not sequence homology) to pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK-phosphonopyruvate complex and the structures of two complexes of pyruvate kinase, one with Mg(2+)-bound phospholactate and the other with Mg(2+)-oxalate and ATP, revealed that the two enzymes share some key features that facilitate common modes of substrate binding. There are also important structural differences; most notably, the machinery for acid/base catalysis is different.

Amino Acid Substitution↗
PubMed1976

Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet.

1. Pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads was measured by using pig heart pyruvate dehydrogenase [32P]phosphate. About 80% was found to be extramitochondrial and therefore probably not directly concerned with the regulation of pyruvate dehydrogenase activity. The extramitochondrial activity was sensitive to activation by Ca2+, but perhaps less sensitive than the mitochondrial activity.

Adipose Tissue↗
PubMed2002

Interaction between the individual isoenzymes of pyruvate dehydrogenase kinase and the inner lipoyl-bearing domain of transacetylase component of pyruvate dehydrogenase complex.

Protein-protein interactions play an important role in the regulation of enzymic activity of pyruvate dehydrogenase kinase (PDK). It is generally believed that the binding of PDK to the inner lipoyl-bearing domain L2 of the transacetylase component E2 of pyruvate dehydrogenase complex largely determines the level of kinase activity. In the present study, we characterized the interaction between the individual isoenzymes of PDK (PDK1-PDK4) and monomeric L2 domain of human E2, as well as the effect of this interaction on kinase activity. It was found that PDK isoenzymes are markedly different with respect to their affinities for L2. PDK3 demonstrated a very tight binding, which persisted during isolation of PDK3-L2 complexes using size-exclusion chromatography. Binding of PDK1 and PDK2 was readily reversible with the apparent dissociation constant of approx. 10 microM for both isoenzymes. PDK4 had a greatly reduced capacity for L2 binding (relative order PDK3>PDK1=PDK2>PDK4). Monomeric L2 domain alone had very little effect on the activities of either PDK1 or PDK2. In contrast, L2 caused a 3-fold increase in PDK3 activity and approx. 37% increase in PDK4 activity. These results strongly suggest that the interactions between the individual isoenzymes of PDK and L2 domain are isoenzyme-specific and might be among the major factors that determine the level of kinase activity of particular isoenzyme towards the pyruvate dehydrogenase complex.

Animals↗
PubMed1999

Nematode pyruvate dehydrogenase kinases: role of the C-terminus in binding to the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex.

Pyruvate dehydrogenase kinases (PDKs) from the anaerobic parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans were functionally expressed with hexahistidine tags at their N-termini and purified to apparent homogeneity. Both recombinant PDKs (rPDKs) were dimers, were not autophosphorylated and exhibited similar specific activities with the A. suum pyruvate dehydrogenase (E1) as substrate. In addition, the activities of both PDKs were activated by incubation with PDK-depleted A. suum muscle pyruvate dehydrogenase complex (PDC) and were stimulated by NADH and acetyl-CoA. However, the recombinant A. suum PDK (rAPDK) required higher NADH/NAD+ ratios for half-maximal stimulation than the recombinant C. elegans PDK (rCPDK) or values reported for mammalian PDKs, as might be predicted by the more reduced microaerobic mitochondrial environment of the APDK. Limited tryptic digestion of both rPDKs yielded stable fragments truncated at the C-termini (trPDKs). The trPDKs retained their dimeric structure and exhibited substantial PDK activity with the A. suum E1 as substrate, but PDK activity was not activated by incubation with PDK-depleted A. suum PDC or stimulated by elevated NADH/NAD+ or acetyl-CoA/CoA ratios. Direct-binding assays demonstrated that increasing amounts of rCPDK bound to the A. suum PDK-depleted PDC. No additional rCPDK binding was observed at ratios greater than 20 mol of rCPDK/mol of PDC. In contrast, the truncated rCPDK (trCPDK) did not exhibit significant binding to the PDC. Similarly, a truncated form of rCPDK, rCPDK1-334, generated by mutagenesis, exhibited properties similar to those observed for trCPDK. These results suggest that the C-terminus of the PDK is not required for subunit association of the homodimer or catalysis, but instead seems to be involved in the binding of the PDKs to the dihydrolipoyl transacetylase core of the complex.

Acetyltransferases↗
PubMed1999

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

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

Animals↗
PubMed1978

The active site of rabbit muscle pyruvate kinase. Evidence for a site common to the oxalacetate decarboxylase and pyruvate kinase reactions.

Rabbit muscle pyruvate kinase has been shown to catalyze the decarboxylation of oxalacetate (Creighton, D.J. and Rose, I.A. (1976) J. Biol. Chem. 251, 61). Noncovalent and covalent modifiers of the enzyme have been used to assess whether the decarboxylase and kinase reactions take place at a common site. Phosphoenol-alpha-ketobutyrate, an analog of the substrate phosphoenol-pyruvate, inhibits decarboxylase and kinase competitively and with nearly identical Ki values (5.7 micron and 4.8 micron, respectively). Oxalate, an analog of enol-pyruvate, inhibits each competitively and with similar Ki values (11 micron and 4.7 micron, respectively). Both activities are lost in parallel upon reaction with dithionitrobenzoate, which is active-site specific. These results indicate that the two activities share a common site on the enzyme. But, effects of the following modifiers suggest that different amino acid residues at that site participate in the two reactions: phenylalanine inhibition and fructose 1,6-bisphosphate activation are more effective with the decarboxylase; iodoacetamide preferentially inactivates decarboxylase while trinitrobenzenesulfonate preferentially inactivates kinase.

Animals↗
PubMed1999

A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism.

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.

Acetyltransferases↗
PubMed1998

High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis. Implications for substrate activation in pyruvate decarboxylases.

The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 A resolution and refined to a crystallographic R-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the alpha/beta type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.

Amino Acid Sequence↗
PubMed1983

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component.

The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1). The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function.

Amino Acids↗
PubMed1979

Lactate-to-pyruvate or pyruvate-to-lactate assay for lactate dehydrogenase: a re-examination.

The pyruvate-to-lactate assay for determining lactate dehydrogenase (EC 1.1.1.27) can now yield linearity equal to or better than that obtained by the lactate-to-pyruvate assay. In addition, there are significant advantages to the pyruvate-to-lactate reaction: (a) a greater change in absorbance per unit time, which allows more accurate spectrophotometric readout; (b) lower reactant concentrations are required, which substantially reduces the cost per assay; (c) solid reagents are used to prepare the assay solution; and (d) reagent solutions are more stable. However, impurities present in commercial NADH preparations may substantially affect measured lactated dehydrogenase activities; therefore, a Standard Reference Material for NADH is being developed for issuance by the National Bureau of Standards.

L-Lactate Dehydrogenase↗
PubMed1978

Optimal conditions and comparison of lactate dehydrogenase catalysis of the lactate-to-pyruvate and pyruvate-to-lactate reactions in human serum at 25, 30, and 37 degrees C.

We report optimal conditions for assaying highly purified human lactate dehydrogenase isoenzymes with the lactate-to-pyruvate and pyruvate-to-lactate reactions, as they apply to human serum. Interconversion of results between reactions is not practicable. Measurements of lactate dehydrogenase in either reaction direction at 25, 30, or 37 degrees C can be equally reliable if the volume fraction and the resulting deltaA/min is small. However, for interinstrument and interlaboratory comparisons, results from the lactate-to-pyruvate reaction are more reliable.

Buffers↗
PubMed2000

Mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency.

Defects in the pyruvate dehydrogenase (PDH) complex are an important cause of primary lactic acidosis, a frequent manifestation of metabolic disease in children. Clinical symptoms can vary considerably in patients with PDH complex deficiencies, and almost equal numbers of affected males and females have been identified, suggesting an autosomal recessive mode of inheritance of the disease. However, the great majority of PDH complex deficiencies result from mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1). The major factors that contribute to the clinical variation in E1alpha deficiency and its resemblance to a recessive disease are developmental lethality in some males with severe mutations and the pattern of X-inactivation in females. To date, 37 different missense/nonsense and 39 different insertion/deletion mutations have been identified in the E1alpha subunit gene of 130 patients (61 females and 69 males) from 123 unrelated families. Insertion/deletion mutations occur preferentially in exons 10 and 11, while missense/nonsense mutations are found in all exons. In males, the majority of missense/nonsense mutations are found in exons 3, 7, 8 and 11, and three recurrent mutations at codons R72, R263 and R378 account for half of these patients with missense/nonsense mutations (25 of 50). A significantly lower number of females is found with missense/nonsense mutations (25). However, 36 females out of 55 affected patients have insertion/deletion mutations. The total number of female and male patients is thus almost the same, although a difference in the distribution of the type of mutations is evident between both sexes. In many families, the parents of the affected patients were studied for the presence of the PDHA1 mutation. The mutation was never present in the somatic cells of the father; in 63 mothers studied, 16 were carriers (25%). In four families, the origin of the new mutation was determined to be twice paternal and twice maternal.

Amino Acid Sequence↗
PubMed1996

Suppression of beta-oxidation restores pyruvate inhibition of pyruvate dehydrogenase kinase in starved rat heart.

Kinetic behaviour of rat heart pyruvate dehydrogenase kinase (PDHK alpha) was studied in the multi-enzyme complex (PDC) contained in two preparations: mitochondria (mPDC) and a high speed pellet of Triton-extracted tissue (hPDC). Two parameters were evaluated: Vav, related to Vmax, and Fractional Pyruvate Inhibition (FPI). Starvation of rats for 48 h led to a rise in Vav and a fall in FPI. Injection into starved rats of agents which reduce beta-oxidation of fatty acids restored, in succession, FPI and then Vav, of hPDC, to levels found in hPDC from fed animals. In vitro incubation at 30 degrees C of hPDC from starved animals restored FPI, but not Vav to 'fed' values; both were restored during in vitro incubation of mPDC from starved animals within the same time frame as in the in vivo experiments. A sharp increase of FPI, but not Vav, of hPDC from both fed and starved rats was observed in later experiments. This could have been due to differential selection of the two genes for isoenzymes of PDHK alpha proposed by other workers.

Animals↗
PubMed1992

Effects of recombinant monokines on hepatic pyruvate dehydrogenase, pyruvate dehydrogenase kinase, lipogenesis de novo and plasma triacylglycerols. Abolition by prior fasting.

1. The effects of recombinant human tumour necrosis factor alpha (TNF) and murine interleukin-1 alpha (IL-1) on the activation state of the hepatic pyruvate dehydrogenase complex (PDHa), the activity of mitochondrial PDH kinase, hepatic lipogenesis de novo and plasma triacylglycerol (TG) concentrations were studied. 2. Monokine effects depended upon prior nutritional state. In rats fasted for 20 h or 45 h before monokine administration and refeeding (orally or with intravenous glucose), PDHa, TG and hepatic lipogenesis were not increased. In rats fed ad libitum, treatment with TNF plus IL-1 increased the contribution of hepatic lipogenesis to circulating TG to 550% of control values (P = 0.03) and plasma TG concentrations to 159% (P = 0.02), whereas PDHa increased slightly to 120% (P = 0.02) and liver glycogen content fell to 45.8% (P = 0.05) of control values. 3. Intrinsic hepatic PDH kinase activity was not changed by monokine treatment in rats fed ad libitum. 4. The increased lipogenesis de novo showed no correlation (r2 = 0.05, not significant) with hepatic PDHa in individual animals fed ad libitum. 5. In conclusion, these results suggest that monokines increase pyruvate flux through hepatic PDH in vivo in rats fed ad libitum primarily by mechanisms other than covalent modification of PDH. Prior nutritional status exerts a permissive effect for monokine stimulation of PDHa and lipogenesis, consistent with a substrate-mediated action, but the mechanism of this permissive effect remains uncertain.

Animals↗
PubMed1998

Requirements for the adaptor protein role of dihydrolipoyl acetyltransferase in the up-regulated function of the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.

The dihydrolipoyl acetyltransferase (E2 component) is a 60-mer assembled via its COOH-terminal domain with exterior E1-binding domain and two lipoyl domains (L2 then L1) sequentially connected by mobile linker regions. E2 facilitates markedly enhanced function of the pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). Human E2 structures were prepared with only one lipoyl domain (L1 or L2) or with alanines substituted at the sites of lipoylation (Lys-46 in L1 or Lys-173 in L2). The L2 domain and its lipoyl group were shown to be essential for markedly enhanced PDP function and were required for greatly up-regulated PDK function. The complete absence of the L1 domain reduced the enhancements of both of these activities but not the maximal effector-stimulated PDK activity through acetylation of L2. With nonlipoylated L2 present, lipoylated L1 supported a lesser enhancement in PDK function with significant stimulation upon acetylation of L1. Prevention of L1 lipoylation in K46AE2 removed this competitive L1 role and enhanced L2-facilitated PDK activity beyond that of native E2 when PDK activity was measured in the absence or in the presence of stimulatory effectors. Thus, the E2-L2 domain has a paramount role in facilitating enhanced PDK and PDP function but inclusion of E2-L1 domain, even in a noninteracting (nonlipoylated) form, contributes to the marked elevation of these activities.

Acetyltransferases↗
PubMed1992

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.

Acetyltransferases↗