Solvent isotope effect on the reaction catalysed by the pyruvate dehydrogenase complex from Escherichia coli

The pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide. The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction. The pH profile and, accordingly, the delta pK1 and delta pK2 values were shifted by 0.6 units to higher pL values. A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0%. This is an indication for a single proton transfer. It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1' nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue. The proton of its carboxylic group exchanges very fast with deuterons of the solvent.     

A transition state analogue for two pyruvate metabolizing enzymes, lactate dehydrogenase and alanine dehydrogenase.

The synthesis of 5-(2-oxalylethyl)-NADH, a reduced nicotinamide adenine dinucleotide (NADH) derivate with pyruvate covalently attached to the 5 position of the dihydronicotinamide ring over an additional methylene group has been described previously (Trommer, W.E., Blume, H., and Kapmeyer, H. (1976) Justus Liebigs Ann. Chem., 848). In the presence of lactate dehydrogenase, the dihydropyridine ring of this coenzyme-substrate analogue is oxidized and the carbonyl function of the side chain is reduced to the corresponding L-hydroxy derivative with a maximum velocity of 1/3000 of the natural reaction. This reaction is intramolecular as shown by competition experiments with pyruvate. 5-(2-oxalylethyl)-NADH (pyr-NADH) appears to be a true transition state analogue, proving its postulated structure. Pyr-NADH is high specific for this enzyme as demonstrated by the facts that (1) D-lactate dehydrogenase does not catalyze the intramolecular redox reaction, although the substrate moiety of pyr-NADH is reduced in the presence of NADH; (2) when tested with malate dehydrogenase, alcohol dehydrogenase, glyceraldehyde phosphate dehydrogenase,glycerate dehydrogenase, and glycerol dehydrogenase pyr-NADH is not even oxidized in the presence of the corresponding substrates. However, a great similarity between the transition states of the reduction of pyruvate catalyzed by lactate dehydrogenase and alanine dehydrogenase could be shown. Alanine dehydrogenase catalyzes the intramolecular redox reaction as well. In the presence of ammonium ions, pyr-NADH is transformed to 5-(3-carboxyl-3-aminopropyl)-NAD+.     

Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding

l-Alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to l-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 Å resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16° toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.     

Binding of thiamin thiazolone pyrophosphate to mammalian pyruvate dehydrogenase and its effects of kinase and phosphatase activities.

The pyruvate dehydrogenase component of the bovine kidney pyruvate dehydrogenase complex has two thiamin-PP binding sites per α₂β₂ tetramer. Titration of these binding sites with the transition state analog, thiamin thiazolone pyrophosphate, strongly inhibits phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase and ATP. The analog has little effect, if any, on dephosphorylation of phosphorylated pyruvate dehydrogenase by pyruvate dehydrogenase phosphatase. Phosphorylation of pyruvate dehydrogenase inactivates the enzyme, but does not significantly affect the thiamin-PP binding sites. It appears that phosphorylation produces a conformational change in pyruvate dehydrogenase that displaces a catalytic group (or groups) at the active center.     

Cyclopropylglyoxylate as a mechanistic probe of thiamine pyrophosphate dependent pyruvate-metabolizing enzymes

Four pyruvate-decarboxylating enzymes with thiamine pyrophosphate (TPP) cofactors catalyze the decarboxylation of the cyclopropyl substrate analog cyclopropylglyoxylate. Pyruvate: ferredoxin oxidoreductase, an archaebacterial enzyme which catalyzes oxidation of the hydroxyethyl-TPP (HETPP) intermediate by two one-electron transfers to an iron-sulfur center, generates the coenzyme A thioester of cyclopropylcarboxylic acid. A long-lived free radical, HETPP is thought to be an intermediate in the pyruvate to acetyl-CoA conversion; however, cleavage of the cyclopropyl ring was not detected. Pyruvate decarboxylase, pyruvate oxidase, and pyruvate dehydrogenase also generate the corresponding cyclopropyl products. The applicability of cyclopropyl substrate analogs as indicators of free-radical enzyme mechanisms is discussed in light of these results.     

Regulation of heart muscle pyruvate dehydrogenase kinase

1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [(32)P]phosphate from [gamma-(32)P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100mum) and cyclic 3':5'-nucleotides (at 10mum) had no significant effect on kinase activity. 3. The K(m) for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76mum. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The K(m) for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9-25.4mum. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The K(m) for pyruvate in the pyruvate dehydrogenase reaction was 35.5mum. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25-500mum. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10mum) the kinase activity was enhanced by low concentrations of pyruvate (25-100mum) and inhibited by a high concentration (500mum). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms (14)CO(2) from [1-(14)C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg(2+) (15mm) and by Ca(2+) (10nm-10mum) at low Mg(2+) (0.15mm) but not at high Mg(2+) (15mm).     

Production of a novel tryptophan analog, beta-1-indazole-L-alanine with tryptophan synthase of Escherichia coli

The tryptophan synthase alpha 2 beta 2 complex from Escherichia coli has been found to catalyze the beta-replacement reaction of L-serine with indazole, an indole analog which has a nitrogen atom at the 2-position (pyrazole ring). The reaction product was isolated and identified as beta-indazolealanine by mass spectrometric, elemental and NMR analyses. Careful assignment of 1H- and 13C-signals with several NMR techniques revealed that the beta-carbon of the product alanine moiety was bound to the 1-N-position of the indazole ring. This is the first example of the beta-replacement reaction catalyzed by tryptophan synthase occurring at any other position than the 3-position of indole analogs.     

Regulatory properties of the pyruvate dehydrogenase complex from Escherichia coli. Studies on the thiamin diphosphate-dependent lag phase

The pyruvate dehydrogenase complex from Escherichia coli shows an appreciable lag phase (tau) of some minutes when its overall reaction rate was tested with very limiting amounts of thiamin diphosphate. tau depends on the concentration of thiamin diphosphate in a nonlinear fashion. Sodium diphosphate, a competitive inhibitor with respect to thiamin diphosphate (Ki = 5.2 . 10(-4) M) prolongs the lag, while the strongly binding transition state analog thiamin thiazolone diphosphate has no effect. tau is independent of the enzyme concentration, thus no dissociation-association step is involved. Incubation of the pyruvate dehydrogenase complex with thiamin diphosphate, Mg2+, and pyruvate leads to a shortening of the lag phase, as well as to a decrease of the intrinsic tryptophan fluorescence in a time-dependent process, which evinces the same characteristics as tau. Dependence of pyruvate, as well as of the substrate analog methylacetylphosphonate, can be established by measurements of fluorescence quenching, thus ruling out an essential role of hydroxyethyl thiamin diphosphate in the process reflected by the lag phase. The results demonstrate that the lag phase is induced after the binding of both thiamin diphosphate . Mg2+ and pyruvate to the catalytic site to form a ternary enzyme complex, which undergoes subsequently a slow conformational change to an active enzyme form. This change is confined to single subunits, and no interactions between neighboring monomers could be observed. A model is proposed to describe the mechanism represented by the lag phase.     

Pyruvate is a by-product of catalysis by ribulosebisphosphate carboxylase/oxygenase

Pyruvate is a minor product of the reaction catalyzed by ribulosebisphosphate carboxylase/oxygenase from spinach leaves. Labeled pyruvate was detected, in addition to the major labeled product, 3-phosphoglycerate, when 14CO2 was the substrate. Pyruvate production was also measured spectrophotometrically in the presence of lactate dehydrogenase and NADH. The Km for CO2 of the pyruvate-producing activity was 12.5 microM, similar to the CO2 affinity of the 3-phosphoglycerate-producing activity. No pyruvate was detected by the coupled assay when ribulose 1,5-bisphosphate was replaced by 3-phosphoglycerate or when the carboxylase was inhibited by the reaction-intermediate analog, 2'-carboxyarabinitol 1,5-bisphosphate. Therefore, pyruvate was not being produced from 3-phosphoglycerate by contaminant enzymes. The ratio of pyruvate produced to ribulose bisphosphate consumed at 25 degrees C was 0.7%, and this ratio was not altered by varying pH or CO2 concentration or by substituting Mn2+ for Mg2+ as the catalytically essential metal. The ratio increased with increasing temperature. Ribulose-bisphosphate carboxylases from the cyanobacterium Synechococcus PCC 6301 and the bacterium Rhodospirillum rubrum also catalyzed pyruvate formation and to the same extent as the spinach enzyme. When the reaction was carried out in 2H2O, the spinach carboxylase increased the proportion of its product partitioned to pyruvate to 2.2%. These observations provide evidence that the C-2 carbanion form of 3-phosphoglycerate is an intermediate in the catalytic sequence of ribulose-bisphosphate carboxylase. Pyruvate is formed by beta elimination of a phosphate ion from a small portion of this intermediate.     

The nature of the substrate inhibition in lactate dehydrogenases as studied by a spin-labeled derivative of NAD.

The formation of the ternary complex of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from pig heart and skeletal muscle with the adduct of pyruvate to NAD", spin-labeled at N6 was studied by ultraviolet spectroscopy and ESR techniques. According to ultraviolet measurements we found identical binding characteristics for the natural coenzyme and its spin-labeled analog. The rate by which the ESR signal of free spin-labeled NAD+ decreased upon addition of pyruvate to the binary complexes was substantially different in the two isozymes. With the heart type an initial drop followed by a further linear decrease, zero order in the enzyme and coenzyme concentration was observed. In case of the skeletal muscle isozyme no immediate reaction and a first order process occurred. The initial reaction can be attributed to a non-covalent enzyme/spin-labeled NAD+/pyruvate complex with a dissociation constant for pyruvate of 11 +/- 1 mM, thus explaining the well-known substrate inhibition in the heart isozyme above 2 mM pyruvate. The further reaction is then determined by the buffer dependent enolization of pyruvate. In the muscle isozyme formation of the covalent adduct is not assisted by prior binding of pyruvate in a non-covalent ternary complex and therefore the rate depends on the binary complex concentration.     

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...     

The pyruvate-dehydrogenase complex from Azotobacter vinelandii.

The pyruvate dehydrogenase complex from Axotobacter vinelandii was isolated in a five-step procedure. The minimum molecular weight of the pure complex is 600,000, as based on an FAD content of 1.6 nmol-mg protein-1. The molecular weight is 1.0-1.2 X 10(6), indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr89,000) and lipoamide dehydrogenase (Mrmonomer 56,000) two active transacetylase isoenzymes are present with molecular weight on the gel 82,000 and 59,000 but probably actually lower. The pure complex has a specific activity of the pyruvate-NAD+ reductase (overall) reaction of 10 units-mg protein-1 at 25 degrees C. The partial reactions have the following specific activities in units-mg protein-1 at 25 degrees C under standard conditions: pyruvate-K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor. Under aerobic conditios pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 mumol 02-min-1-mg-1, Km(pyruvate)0.3 mM. The kinetics of this reaction shows a linear 1/velocity-1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity is stimulated by AMP and sulphate and is inhibited by acetyl-CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.     

Pyruvate dehydrogenase and 3-fluoropyruvate: chemical competence of 2-acetylthiamin pyrophosphate as an acetyl group donor to dihydrolipoamide

The pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex catalyzes the decomposition of 3-fluoropyruvate to CO2, fluoride anion, and acetate. Acetylthiamin pyrophosphate (acetyl-TPP) is an intermediate in this reaction. Incubation of the pyruvate dehydrogenase complex with 3-fluoro[1,2-14C]pyruvate, TPP, coenzyme A (CoASH), and either NADH or pyruvate as reducing systems leads to the formation of [14C]acetyl-CoA. In this reaction the acetyl group of acetyl-TPP is partitioned by transfer to both CoASH (87 +/- 2%) and water (13 +/- 2%). When the E1 component is incubated with 3-fluoro[1,2-14C]pyruvate, TPP, and dihydrolipoamide, [14C]acetyldihydrolipoamide is produced. The formation of [14C]acetyldihydrolipoamide was examined as a function of dihydrolipoamide concentration (0.25-16 mM). A plot of the extent of acetyl group partitioning to dihydrolipoamide as a function of 1/[dihydrolipoamide] showed 95 +/- 2% acetyl group transfer to dihydrolipoamide when dihydrolipoamide concentration was extrapolated to infinity. It is concluded that acetyl-TPP is chemically competent as an intermediate for the pyruvate dehydrogenase complex catalyzed oxidative decarboxylation of pyruvate.     

Inhibition of lactate glucogneogenesis in rat kidney by dichloroacetate.

1. Sodium dichloroacetate (1mM) inhibited glucose production from L-lactate in kidney-cortex slices from fed, starved or alloxan-diabetic rates. In general gluconeogenesis from other substrates was no inhibited. 2. Sodium dichloracetate inhibited glucose production from L-lactate but no from pyruvate in perfused isolated kidneys from normal or alloxan-diabetic rats. 3. Sodium dichloroacetate is an inhibitor of the pyruvate dehydrogenase kinase reaction and it effected conversion of pyruvate dehydrogenase into its its active (dephosphorylated) form in kidney in vivo. In general, pyruvate dehydrogenase was mainly in the active form in kidneys perfused or incubated with L-lactate and the inhibitory effect of dichloroacetate on glucose production was not dependent on activation of pyruvate dehydrogenase. 4. Balance data from kidney slices showed that dichloroacetate inhibits lactate uptake, glucose and pyruvate production from lactate, but no oxidation of lactate. 5. The mechanism of this effect of dichloroactetate on glucose production from lactate has not been fully defined, but evidence suggests that it may involve a fall in tissue pyruvate concentration and inhibition of pyruvate carboxylation.     

Hydrolysis of phosphoenolpyruvate catalyzed by phosphoenolpyruvate carboxylase from Zea mays.

In addition to the normal carboxylation reaction, phosphoenolpyruvate carboxylase from Zea mays catalyzes a HCO3(-)-dependent hydrolysis of phosphoenolpyruvate to pyruvate and Pi. Two independent methods were used to establish this reaction. First, the formation of pyruvate was coupled to lactate dehydrogenase in assay solutions containing high concentrations of L-glutamate and aspartate aminotransferase. Under these conditions, oxalacetic acid produced in the carboxylation reaction was efficiently transaminated, and decarboxylation to form spurious pyruvate was negligible. Second, sequential reduction of oxalacetate and pyruvate was achieved by initially running the reaction in the presence of malate dehydrogenase with NADH in excess over phosphoenolpyruvate. After the reaction was complete, lactate dehydrogenase was added, thus giving a measure of pyruvate concentration. At pH 8.0 in the presence of Mg2+, the rate of phosphoenolpyruvate hydrolysis was 3-7% of the total reaction rate. The hydrolysis reaction catalyzed by phosphoenolpyruvate carboxylase was strongly metal dependent, with rates decreasing in the order Ni2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. These results suggest that the active site metal ion binds to the enolate oxygen, thus stabilizing the proposed enolate intermediate. The more stable the enolate, the less reactive it is toward carboxylation and the greater the opportunity for hydrolysis.     

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.     

An enzymatic cycling method for 3-acetylpyridine adenine dinucleotide to increase the sensitivity of enzymatic methods which employ this NAD analog

The NAD analog 3-acetylpyridine adenine nucleotide (APAD), because of its higher oxidation potential, has proven useful for the direct enzymatic measurement of such compounds as lactate, malate, glutamate, etc., for which the equilibrium with NAD+ as oxidant is unfavorable. An enzymatic cycling method which is capable of increasing the sensitivity of such reactions 10,000-fold or more is described. The APADH produced in the original stoichiometric reaction is used to catalyze a cycling reaction that employs lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37) to generate (from lactate plus oxalacetate) very large quantities of pyruvate and malate. After the cycling step, the malate formed is measured with NAD+ and with malate dehydrogenase, plus aspartate aminotransferase, and oxaloacetate to pull this indicator reaction to completion. The application of this cycling method is illustrated by analysis of malate in the range 1 to 10 pmol.     

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.     

Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.     

Substrate-induced structural changes of the pyruvate dehydrogenase multienzyme complex

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