Balázs Sümegi

University of Pécs, Fuenfkirchen, Baranya county, Hungary

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Publications (9)28.32 Total impact

  • Balázs Sümegi, József Batke, Zoltán Porpáczy
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    ABSTRACT: 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.
    Archives of Biochemistry and Biophysics 03/1985; 236(2):741-52. DOI:10.1016/0003-9861(85)90680-0 · 3.04 Impact Factor
  • Zoltan Porpaczy, Balázs Sümegi, István Alkonyi
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    ABSTRACT: The kinetic parameters of the individual reaction of pig heart alpha-ketoglutarate dehydrogenase complex, succinate thiokinase and the alpha-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system were studied. The KCoAm of alpha-ketoglutarate dehydrogenase complex and the K-succinyl CoAm of succinate thiokinase decreased in the coupled system when compared to those of the individual enzyme reactions. This phenomenon can be explained by the interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. By means of poly(ethylene glycol) precipitation, ultracentrifugation and gel chromatography we were able to detect a physical interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. Of the seven investigated proteins only succinate thiokinase showed association with alpha-ketoglutarate dehydrogenase complex. On the other hand, succinate thiokinase did not associate with other high molecular weight mitochondrial enzymes such as pyruvate dehydrogenase complex and glutamate dehydrogenase. On this basis, the interaction between succinate thiokinase and alpha-ketoglutarate dehydrogenase complex was assumed to be specific. These in vitro data raise the possibility that a portion of the citric acid cycle enzymes exists as a large multienzyme complex in the mitochondrial matrix.
    Biochimica et Biophysica Acta 01/1984; 749(2):172-9. DOI:10.1016/0167-4838(83)90249-2 · 4.66 Impact Factor
  • Balázs Sümegi, István Alkonyi
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    ABSTRACT: In this paper, physicochemical evidence is given for the association between the pyruvate dehydrogenase complex (EC and citrate synthase (EC with two gel chromatographic techniques with poly(ethylene glycol) co-precipitation and with ultracentrifugation. Experiments with active enzyme gel chromatography indicate that citrate synthase also associates with pyruvate dehydrogenase complex in its functioning state. Citrate synthase binds to the isolated transacetylase core of pyruvate dehydrogenase complex, but in the binding to the whole pyruvate dehydrogenase complex the two other components of the complex are also involved. One pyruvate dehydrogenase complex can bind 10-11 citrate synthase dimers, and the dissociation constant is about 5.7-6.0 microM as determined by two independent methods. The association between the pyruvate dehydrogenase complex and citrate synthase raises the possibility of the dynamic compartmentation of acetyl-CoA in the mitochondria which results in the direction of acetyl-CoA from pyruvate towards citrate.
    Biochimica et Biophysica Acta 01/1984; 749(2):163-71. DOI:10.1016/0167-4838(83)90248-0 · 4.66 Impact Factor
  • B Sümegi, I Alkonyi
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    ABSTRACT: In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.
    European Journal of Biochemistry 12/1983; 136(2):347-53. · 3.58 Impact Factor
  • Balázs Sümegi, István Alkonyi
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    ABSTRACT: The phenomenon of paracatalytic inactivation has been demonstrated and characterized with the pig heart pyruvate dehydrogenase complex. The enzyme became progressively inactive when it was preincubated in the presence of pyruvate, thiamine pyrophosphate, and extrinsic oxidative agent, 2,6-dichloroindophenol. Not only the overall reaction of enzyme complex but the individual reactions catalyzed by enzyme components, pyruvate dehydrogenase, and dihydrolipoamide acetyltrasferase, were reduced after preincubation. The observed inactivation is due to two factors: (i) covalent incorporation of the pyruvate atoms, and (ii) formation of a thiamine pyrophosphate analog. (i) A covalent incorporation of radioactivity from [2-14C]pyruvate into the enzyme complex, which was proportional to the inhibition of overall reaction was observed. Seventy-eight percent of the radioactivity incorporated into the dihydrolipoamide transacetylase but not into the lipoic acid. Thus, modification of the dihydrolipoamide acetyltransferase component can be attributed to the covalent incorporation of pyruvate atoms. (ii) During inactivation, the enzyme-bound thiamine pyrophosphate underwent a modification resulting in the formation of a thiamine pyrophosphate analog, presumably thiamine thiazolone pyrophosphate, which inhibited exclusively the pyruvate dehydrogenase component.
    Archives of Biochemistry and Biophysics 07/1983; 223(2):417-24. DOI:10.1016/0003-9861(83)90605-7 · 3.04 Impact Factor
  • Balázs Sümegi, László Gyócsi, Alkonyi
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    ABSTRACT: Pig heart pyruvate dehydrogenase complex (pyruvate: lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC catalyzes the decomposition of diacetyl to acetate, acetyl-CoA and produces reduced NAD+ with 1:1:1 stoichiometry. The reaction rate with diacetyl is approximately 7.1% of that with pyruvate. The Km value for diacetyl was found to be 0.46 mM. Acetoin and acetaldehyde inhibit the pyruvate dehydrogenase-catalyzed reaction of diacetyl with Ki values of 0.91 mM and 0.48 mM, respectively. Inhibition patterns show that they are competitive inhibitors versus diacetyl. Acetate, as product, does not inhibit the enzymatic decomposition of diacetyl. Diacetyl is not only an alternative substrate in the pyruvate dehydrogenase reaction, but a competitive inhibitor versus pyruvate with a Ki value of 0.43 mM.
    Biochimica et Biophysica Acta 08/1982; 705(1):70-5. DOI:10.1016/0167-4838(82)90337-5 · 4.66 Impact Factor
  • Balázs Sümegi, László Gyócsi, István Alkonyi
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    ABSTRACT: Kinetic studies of the individual reaction of pig heart pyruvate dehydrogenase complex (pyruvate dehydrogenase (pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC; dihydrolipoamide reductase(NAD+) (NADH:lipoamide oxidoreductase, EC; dihydrolipoamide acetyltransferase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC, citrate synthase (citrate oxaloacetate-lyase (pro-3S-CH2COO- leads to acetyl-CoA), EC and the pyruvate dehydrogenase complex-citrate synthase coupled system show that the KmCoA value of pyruvate dehydrogenase complex and KmCoASAc value of citrate synthase decrease in the coupled system when compared to those in the individual enzyme reactions. The explanation for this interaction may be an association between the two enzymes. When it was centrifuged with 150 000 x g for 140 min, 30% of the citrate synthase sedimented in the presence of the pyruvate dehydrogenase complex, while no sedimentation was observed in the absence of the pyruvate dehydrogenase complex. Sedimentation of cytoplasmic malate dehydrogenase, phosphotransacetylase, hemoglobin and Blue albumin were negligible under the same condition. In gel chromatography experiments a significant peak of citrate synthase activity co-migrated with the pyruvate dehydrogenase complex peak. This observation also suggests the possible association of two enzymes.
    Biochimica et Biophysica Acta 01/1981; 616(2):158-66. DOI:10.1016/0005-2744(80)90134-5 · 4.66 Impact Factor
  • I Alkonyi, L Gyócsi, B Sümegi
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    ABSTRACT: At low thiamine pyrophosphate concentrations the time-course of the reaction catalyzed by mammalian pyruvate dehydrogenase complex shows a lag period of some minutes when the reaction is started by either enzyme, pyruvate or thiamine pyrophosphate. However, started by CoASH or NAD+, the lag period disappears. An increase in enzyme concentration to 25 mU/ml causes a concomitant shortening of the duration of the lag period (tau), while above this value tau is independent of the enzyme concentration. An increase in thiamine pyrophosphate concentration decreases the value of tau and the lag period vanishes at infinite thiamine pyrophosphate concentration. It is suggested that both isomerization and aggregation-dissociation reactions may play an improtant role in the development of the lag period of pyruvate dehydrogenase complex.