Continuous coupled assay for 5-aminolevulinate synthase.
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5-Aminolevulinate Synthetase↗
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Mechanism of the action and of the allosteric inhibition of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (tyrosine-sensitive) of Escherichia coli W.
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Aldehyde-Lyases↗
[Involvement of tau protein kinase in amyloid-beta-induced neurodegeneration].
Histopathological features of Alzheimer's disease (AD) include extracellular deposits of amyloid beta (A beta) fibrils in the cores of senile plaques, intracellular neurofibrillary tangles (NFT) which are composed of paired helical filaments (PHF), and neuronal cell loss. The main component of PHF is highly phosphorylated tau protein. We identified a protein kinase converting normal tau into a PHF-like state. The kinase is tau protein kinase (TPK) I/glycogen synthase kinase (GSK)-3 beta. Using a neuronal cell culture system as an AD model, it was recognized that TPK I/GSK-3 beta plays a central role in AD pathology. We hypothesize that A beta-induced neuronal cell death occurs by the following mechanism. A beta inactivates PI3-kinase and activates TPK I/GSK-3 beta, which in turn phosphorylates and inactivates both tau and pyruvate dehydrogenase (PDH). After the ability of tau to promote microtubule assembly is diminished by phosphorylation, soluble tau molecules aggregate into PHF by an unknown mechanism. Destabilization of microtubule arrays causes inhibition of axonal transport and accumulation of amyloid precursor protein (APP). Phosphorylation of PDH inhibits the reaction converting pyruvate to acetyl-CoA, resulting in inhibition of energy metabolism and a decrease in acetylcholine, both of which are also characteristics of AD. These changes may lead to neuronal cell death.
Alzheimer Disease↗
A continuous method for enzymatic assay of sucrose synthase in the synthetic direction.
An appropriate method was developed for the continuous assay of sucrose synthase (SS) (EC 2.4.1.13) by spectrophotometry. The uridine 5'-diphosphate derived from sucrose synthesis was stoichiometrically coupled to oxidation of beta-nicotinamide adenine dinucleotide by the enzymes nucleoside-5'-diphosphate kinase (NDPK), pyruvate kinase, and lactate dehydrogenase. Utilization of crude extracts led to a complete masking of SS assay by adenylate kinase, adenosine 5'-triphosphatase (ATPase), and phosphoenolpyruvate phosphatase found in the crude extracts. These interfering enzymes were mostly removed from the crude extracts by using a combination of gel filtration, centrifugation through a selectively permeable membrane (Biomax-100 Ultrafree centrifugal device), and inhibition by the addition of K(2)HPO(4) to the assay buffer. Sensitivity of the SS assay was significantly increased by the inclusion of NDPK and ATP, which are essential to the reaction in the coupling system.
Glucosyltransferases↗
Genes involved in meso-diaminopimelate synthesis in Bacillus subtilis: identification of the gene encoding aspartokinase I.
Thermosensitive mutants of Bacillus subtilis deficient in peptidoglycan synthesis were screened for mutations in the meso-diaminopimelate (LD-A2pm) metabolic pathway. Mutations in two out of five relevant linkage groups, lssB and lssD, were shown to induce, at the restrictive temperature, a deficiency in LD-A2pm synthesis and accumulation of UDP-MurNAc-dipeptide. Group lssB is heterogeneous; it encompasses mutations that confer deficiency in the deacylation of N-acetyl-LL-A2pm and accumulation of this precursor. Accordingly, these mutations are assigned to the previously identified locus dapE. Mutations in linkage group lssD entail a thermosensitive aspartokinase 1. Therefore, they are most likely to affect the structural gene of this enzyme, which we propose to designate dapG. Mutation pyc-1476, previously reported to affect the pyruvate carboxylase, was shown to confer a deficiency in aspartokinase 1, not in the carboxylase, and to belong to the dapG locus, dapG is closely linked to spoVF, the putative gene of dipicolinate synthase. In conclusion, mutations affecting only two out of eight steps known to be involved in LD-A2pm synthesis were uncovered in a large collection of thermosensitive mutants obtained by indirect selection. We propose that this surprisingly restricted distribution of the thermosensitive dap mutations isolated so far is due to the existence, in each step of the pathway, of isoenzymes encoded by separate genes. The biological role of different aspartokinases was investigated with mutants deficient in dapE and dapG genes. Growth characteristics of these mutants in the presence of various combinations of aspartate family amino acids allow a reassessment of a metabolic channel hypothesis, i.e. the proposed existence of multienzyme complexes, each specific for a given end product.
Aspartate Kinase↗
Inhibition of calcineurin increases monocarboxylate transporters 1 and 4 protein and glycolytic enzyme activities in rat soleus muscle.
1. The present study was designed to examine the role of calcineurin in muscle metabolic components by the administration of the specific calcineurin inhibitor cyclosporine A (CsA) to rats. 2. Male Wistar rats were divided into either a CsA-treated group (CT) or a vehicle-treated group (VT). Cyclosporine A was administered subcutaneously to rats at a rate of 25 mg/kg bodyweight per day for 10 successive days. Thereafter, changes in muscle enzyme activities and glucose transporter (GLUT)-4 and monocarboxylate transporter (MCT)-1 and MCT-4 proteins in the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles were examined. 3. There was a significant increase in MCT-1 and MCT-4 proteins in the soleus muscle in the CT group, but not in the EDL muscle. The activities of hexokinase, pyruvate kinase and lactate dehydrogenase in the soleus muscle also increased significantly in the CT group, but a similar increase in enzyme activity was not seen in EDL muscle. The activities of citrate synthase or malate dehydrogenase and the GLUT-4 protein content were not altered by CsA treatment in either the soleus or EDL muscles. 4. These results seem to imply that calcineurin negatively regulates the components of glucose/lactate metabolism, except for GLUT-4, especially in slow-twitch muscle.
Animals↗
Small-angle X-ray scattering on malate synthase from baker's yeast. The native substrate-free enzyme and enzyme-substrate complexes.
Malate synthase from baker's yeast has been investigated in solution by the small-angle X-ray scattering technique. Size, shape and structure of the native substrate-free enzyme and of various enzyme-substrate complexes have been determined. As the enzyme was found to be rather unstable against X-rays, several precautions as well as sophisticated evaluation procedures had to be adopted to make sure that the results were not influenced by radiation damage. These included use of low primary intensity, short time of measurement, the presence of high concentrations of dithiothreitol, combined use of the conventional slit-collimation system and the new cone-collimation system. 1. For the native substrate-free enzyme the following molecular parameters could be established: radius of gyration R = 3.96 +/- 0.02 nm, maximum particle diameter D = 11.2 +/- 0.6 nm, radius of gyration of the thickness Rt = 1.04 +/- 0.04 nm, molecular weight Mr = 187000 +/- 3000, correlation volume Vc = 338 +/- 5 nm3, hydration x = 0.35 +/- 0.02 g/g, mean intersection length - l = 5.0 +/- 0.2 nm. Comparison of the experimental scattering curve with theoretical curves for various models showed that the enzyme is equivalent in scattering to an oblate ellipsoid of revolution rather than to a circular cylinder. The semiaxes of this ellipsoid are a = b = 6.06 nm and c = 2.21 nm. Thus with an axial ratio of about 1:0.36 the enzyme is of very anisometric shape. 2. Binding of the substrates (acetyl-CoA, glyoxylate) or the substrate analogue pyruvate causes slight structural changes of the enzyme. These changes are reflected mainly by a slight decrease of the radius of gyration (0.3--1.3%, as established both with the slit-smeared and the desmeared curves). Concomitantly there occurs a decrease of the maximum particle diameter and an increase of the radius of gyration of the thickness. These changes imply an increase of the axial ratio by 2.2--6.9%, i.e. substrate binding induces a decrease of anisometry. While the particle volume appears to be unchanged on binding glyoxylate or its analogue pyruvate, binding of acetyl-CoA causes slight changes of this parameter. In a similar manner the binding of acetyl-CoA leads to a slight enhancement of the molecular weight; this increase corresponds to the binding of 2.7 +/- 1 molecules of acetyl-CoA.
Acetyl Coenzyme A↗
Lactic acidosis and mitochondrial dysfunction in two children with peroxisomal disorders.
Mitochondrial myopathies and defects in oxidative phosphorylation have been described in some patients with peroxisomal disorders. Although peroxisomes and mitochondria play a role in the beta-oxidation of fatty acids, the metabolic interactions between the two are not well defined. Defects in peroxisomal beta-oxidation are associated with extracellular accumulation of very long-chain fatty acids and may be accompanied by alterations in the intracellular pool of fatty acyl-CoAs, which are known to alter mitochondrial function. This study was initiated to examine alterations in the intracellular pool of acyl-CoAs and mitochondrial function in two children with generalized disorders of peroxisomal function and clinical lactic/pyruvic acidaemia. Fibroblasts were cultured from skin biopsies obtained from one child with neonatal adrenoleukodystrophy (NALD) and another with rhizomelic chondrodysplasia punctata (RCDP). Fibroblast lactate oxidation was significantly inhibited in NALD by 76% and RCDP by 92% compared to control values of 1.9 +/- 0.1 nmol/min per mg protein. Pyruvate dehydrogenase (PDH) (mean +/- SEM; activity nmol/min per mg protein) was: NALD 0.55 +/- 0.02 (p < 0.01), RCDP 0.44 +/- 0.02 (P < 0.01), and controls 0.83 +/- 0.02. The acid-insoluble (long-chain and very long-chain) acyl-CoA levels (mean +/- SEM; pmol/mg protein) were: NALD 129 +/- 69 (p < 0.01), RCDP 65 +/- 15 (p < 0.05), and control 45 +/- 7. These two patients with generalized peroxisomal disorders exhibited an increase in intracellular acyl-CoA species accompanied by decreased PDH activity and clinical lactic/pyruvic acidaemia.
Acidosis, Lactic↗
Modulation of proton pumping efficiency in bacterial ATP synthases.
The ATP synthase in chromatophores of Rhodobacter caspulatus can effectively generate a transmembrane pH difference coupled to the hydrolysis of ATP. The rate of hydrolysis was rather insensitive to the depletion of ADP in the assay medium by an ATP regenerating system (phospho-enol-pyruvate (PEP) and pyruvate kinase (PK)). The steady state values of DeltapH were however drastically reduced as a consequence of ADP depletion. The clamped concentrations of ADP obtained using different PK activities in the assay medium could be calculated and an apparent Kd approximately 0.5 microM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate to the assay medium. The Kd for this effect was about 70 microM. Analogous experiments were performed in membrane fragment from Escherichia coli. In this case, however, the hydrolysis rate was strongly inhibited by Pi, added up to 3 mM. Inhibition by Pi was nearly completely suppressed following depletion of ADP. The Kd's for the ADP and Pi were in the micromolar range and submillimolar range, respectively, and were mutually dependent from the concentration of the other ligand. Contrary to hydrolysis, the pumping of protons was rather insensitive to changes in the concentrations of the two ligands. At intermediate concentrations, proton pumping was actually stimulated, while the hydrolysis was inhibited. It is concluded that, in these two bacterial organisms, ADP and phosphate induce a functional state of the ATP synthase competent for a tightly coupled proton pumping, while the depletion of either one of these two ligands favors an inefficient (slipping) functional state. The switch between these states can probably be related to a structural change in the C-terminal alpha-helical hairpin of the epsilon-subunit, from an extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to a retracted one, in which ATP hydrolysis and proton pumping are loosely coupled.
Adenosine Diphosphate↗
Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant.
Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K(m) values for the conversion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant enzymes, and a 5-fold increase in K(m) for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylglycine (L-VG) (deamination to form alpha-ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. L-VG is both a substrate and a mechanism-based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA from the (R,S) diastereomer of SAM to produce L-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107-112], but catalyzes the formation of ACC to a lesser extent via alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.
Catalytic Domain↗
Bacillus subtilis glutamine synthetase mutants pleiotropically altered in glucose catabolite repression.
Strain SF22, a glutamine-requiring (Gln-) mutant of Bacillus subtilis SMY, is likely to have a mutation in the structural gene for glutamine synthetase, since this strain synthesized 22 to 55% as much glutamine synthetase antigen as did wild-type cells in a 10-min period but had less than 3% of wild-type glutamine synthetase enzymatic activity. The expression of several genes subject to glucose catabolite repression was altered in the Gln- mutant. The induced levels of alpha-glucosidase, histidase, and aconitase were 3.5- to 4-fold higher in SF22 cells than in wild-type cells grown in glucose-glutamine medium, and citrate synthase levels were 8-fold higher in the Gln- mutant than in wild-type cells. The relief of glucose catabolite repression in the Gln- mutant may result from poor utilization of glucose. Examination of the intracellular metabolite pools of cells grown in glucose-glutamine medium showed that the glucose-6-phosphate pool was 2.5-fold lower, the pyruvate pool was 4-fold lower, and the 2-ketoglutarate pool was 2.5-fold lower in the Gln- cells than they were in wild-type cells. Intracellular levels of glutamine were sixfold higher in the Gln- mutant than in wild-type cells. Measurements of enzymes involved in glutamine transport and utilization showed that the elevated pools of glutamine in the Gln- mutant resulted from a threefold increase in glutamine permease and a fivefold decrease in glutamate synthase. The pleiotropic effect of the gln-22 mutation on the expression of several genes suggests that either the glutamine synthetase protein or its enzymatic product, glutamine, is involved in the regulation of several metabolic pathways in B. subtilis.
Bacillus subtilis↗
The separation of rat liver serine dehydratase and cystathionine synthase.
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Acetone↗
Selenocysteine synthase from Escherichia coli. Analysis of the reaction sequence.
The product of the selA gene, selenocysteine synthase, is a pyridoxal 5-phosphate-containing enzyme which catalyzes the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA). Reduction of the aldimine group of pyridoxal 5-phosphate inactivates the enzyme. When reacted with seryl-tRNA(Sec UCA) as sole substrate, pyruvate (and possibly also ammonia) is released; in the presence of a high concentration of potassium borohydride, alanyl-tRNA(Sec UCA) is formed from seryl-tRNA(Sec UCA). These results support the notion that the formyl group of pyridoxal phosphate forms a Schiff base with the alpha-amino group of L-serine with the subsequent 2,3-elimination of a water molecule and the generation of an aminoacrylyl-tRNA(Sec UCA) intermediate. ATP is not required for this reaction step, but it is necessary for the conversion of aminoacrylyl-tRNA into selenocysteyl-tRNA(Sec UCA) which, in addition, requires the SELD protein and reduced selenium. Selenocysteine synthase forms a stable complex with seryl-tRNA(Sec UCA) with one tRNA molecule bound per two 50-kDa monomers. The enzyme does not interact with serine-inserting tRNA species. Taken together, the results show that biosynthesis of selenocysteine takes place in the enzyme-bound state and involves the dehydration of L-serine esterified to tRNA in a first step formally followed by the 2,3-addition of HSe- which is provided by the SELD protein in an ATP-dependent reaction in the form of a reactive selenium donor molecule.
Autoradiography↗
Stoichiometric model for evaluating the metabolic capabilities of the facultative methylotroph Methylobacterium extorquens AM1, with application to reconstruction of C(3) and C(4) metabolism.
A stoichiometric model of central metabolism was developed based on new information regarding metabolism in this bacterium to evaluate the steady-state growth capabilities of the serine cycle facultative methylotroph Methylobacterium extorquens AM1 during growth on methanol, succinate, and pyruvate. The model incorporates 20 reversible and 47 irreversible reactions, 65 intracellular metabolites, and experimentally-determined biomass composition. The flux space for this underdetermined system of equations was defined by finding the elementary modes, and constraints based on experimental observations were applied to determine which of these elementary modes give a reasonable description of the flux distribution for each growth substrate. The predicted biomass yield, on a carbon atom basis, is 49.8%, which agrees well with the range of published experimental yield measurements (37-50%). The model predicts the cell to be limited by reduced pyridine nucleotide availability during methylotrophic growth, but energy-limited when growing on multicarbon substrates. Mutation and phenotypic analysis was used to explore a previously unknown region of the metabolic map and to confirm the stoichiometry of the pathways in this region used in the metabolic model. Based on genome sequence data and simulation results, three enzymes involved in C(3)-C(4) interconversion pathways were predicted to be mutually redundant: malic enzyme, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate synthase. Insertion mutations in the genes predicted to encode these enzymes were made and these mutants were capable of growing on all substrates tested, confirming the redundancy of these pathways. Likewise, pathway analysis suggests that the TCA cycle enzymes citrate synthase and succinate dehydrogenase are essential for all growth substrates. In keeping with these predictions, null mutants could not be obtained in these genes. Finally, a similar model was developed for the ribulose monophosphate pathway obligate methylotroph Methylobacillus flagellatum KT to compare the efficiency of carbon utilization in the two types of methylotrophic carbon utilization pathways. The predicted yield for this organism on methanol is 65.9%.
Biomass↗
Effect of short-term sprint interval training on human skeletal muscle carbohydrate metabolism during exercise and time-trial performance.
Our laboratory recently showed that six sessions of sprint interval training (SIT) over 2 wk increased muscle oxidative potential and cycle endurance capacity (Burgomaster KA, Hughes SC, Heigenhauser GJF, Bradwell SN, and Gibala MJ. J Appl Physiol 98: 1895-1900, 2005). The present study tested the hypothesis that short-term SIT would reduce skeletal muscle glycogenolysis and lactate accumulation during exercise and increase the capacity for pyruvate oxidation via pyruvate dehydrogenase (PDH). Eight men [peak oxygen uptake (VO2 peak)=3.8+/-0.2 l/min] performed six sessions of SIT (4-7x30-s "all-out" cycling with 4 min of recovery) over 2 wk. Before and after SIT, biopsies (vastus lateralis) were obtained at rest and after each stage of a two-stage cycling test that consisted of 10 min at approximately 60% followed by 10 min at approximately 90% of VO2 peak. Subjects also performed a 250-kJ time trial (TT) before and after SIT to assess changes in cycling performance. SIT increased muscle glycogen content by approximately 50% (main effect, P=0.04) and the maximal activity of citrate synthase (posttraining: 7.8+/-0.4 vs. pretraining: 7.0+/-0.4 mol.kg protein -1.h-1; P=0.04), but the maximal activity of 3-hydroxyacyl-CoA dehydrogenase was unchanged (posttraining: 5.1+/-0.7 vs. pretraining: 4.9+/-0.6 mol.kg protein -1.h-1; P=0.76). The active form of PDH was higher after training (main effect, P=0.04), and net muscle glycogenolysis (posttraining: 100+/-16 vs. pretraining: 139+/-11 mmol/kg dry wt; P=0.03) and lactate accumulation (posttraining: 55+/-2 vs. pretraining: 63+/-1 mmol/kg dry wt; P=0.03) during exercise were reduced. TT performance improved by 9.6% after training (posttraining: 15.5+/-0.5 vs. pretraining: 17.2+/-1.0 min; P=0.006), and a control group (n=8, VO2 peak=3.9+/-0.2 l/min) showed no change in performance when tested 2 wk apart without SIT (posttraining: 18.8+/-1.2 vs. pretraining: 18.9+/-1.2 min; P=0.74). We conclude that short-term SIT improved cycling TT performance and resulted in a closer matching of glycogenolytic flux and pyruvate oxidation during submaximal exercise.
Adult↗
Paradoxical downregulation of the glucose oxidation pathway despite enhanced flux in severe heart failure.
Free fatty acid (FFA) oxidation is depressed in severe heart failure due to reduced activity of mitochondrial fatty acid oxidation enzymes. It is unknown whether the concomitant enhancement in cardiac glucose use is a consequence of reduced FFA oxidation, or also due to potentiation of the carbohydrate oxidative pathway. FFA and glucose oxidation rates were measured in vivo in 9 normal dogs and 9 dogs with pacing-induced heart failure by infusing (3)H-oleate and (14)C-glucose. FFA oxidation was lower (39 +/- 9 vs. 73 +/- 5 nmol min(-1) g(-1)), while glucose oxidation was higher (42 +/- 8 vs. 17 +/- 6 nmol min(-1) g(-1)) in failing compared to normal hearts (P < 0.05). At the end of the in vivo experiment, clamp-frozen biopsies were harvested from the left ventricle. Messenger RNAs encoding for proteins involved in both glucose and fatty acid metabolism, and for citrate synthase, were significantly reduced. Protein expression of GLUT-1 and GLUT-4, and GLUT-4 translocation to the sarcolemma showed no significant differences between the two groups despite a significant reduction in mRNAs with heart failure. GAPDH mRNA, protein expression, and activity were all reduced. The E2 subunit of pyruvate dehydrogenase was decreased both at the mRNA and protein level, with no effect on either fractional or maximal activity. In conclusion, we found either no changes or moderate downregulation of key enzymes of the carbohydrate metabolism in failing hearts, which suggests that the increase in glucose oxidation in vivo was principally due to impaired FFA oxidation and that the maximal myocardial capacity to obtain energy from substrate is globally depressed.
Animals↗
Calculation of the intracellular distribution of acetyl CoA and CoA, based on the use of citrate synthase as an equilibrium enzyme.
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Acetates↗