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PubMed1992

Indole protects tryptophan indole-lyase, but not tryptophan synthase, from inactivation by trifluoroalanine.

Trifluoroalanine is a mechanism-based inactivator of Escherichia coli tryptophan indole-lyase (tryptophanase) and E. coli tryptophan synthase (R. B. Silverman and R. H. Abeles, 1976, Biochemistry 15, 4718-4723). We have found that indole is able to prevent inactivation of tryptophan indole-lyase by trifluoroalanine. The protection of tryptophan indole-lyase by indole exhibits saturation kinetics, with a KD of 0.03 mM, which is comparable to the KI for inhibition of pyruvate ion formation (0.01 mM) and the Km for L-tryptophan synthesis. Fluoride electrode measurements indicate the formation of 28 mol of fluoride ion per mole of enzyme during inactivation of tryptophan indole-lyase, and 121 mol of fluoride ion are formed per mole of enzyme in the presence of 2 mM indole during the same incubation period. 19F NMR spectra of reaction mixtures of tryptophan indole-lyase and trifluoroalanine showed evidence only for fluoride ion formation, in either the absence or the presence of indole, and difluoropyruvic acid was not detected. The partition ratio, kcat/kinact, is estimated to be 9. Tryptophan indole-lyase in the presence of trifluoroalanine exhibits visible absorption peaks at 446 and 478 nm, which decay at the same rate as inactivation. However, in the presence of 1 mM indole and trifluoralanine, tryptophan indole-lyase exhibits a peak only at 420 nm, and the spectra show a gradual increase at 300-310 nm with incubation. In contrast, tryptophan synthase is not protected by indole from inactivation by trifluoroalanine, and the absorption peak at 408 nm for the tryptophan synthase-trifluoroalanine complex is unaffected by indole. These results demonstrate that inactivation of tryptophan indole-lyase occurs via a catalytically competent species, probably the beta,beta-difluoro-alpha-aminoacrylate intermediate, which can be partitioned from inactivation to products by a reactive aromatic nucleophile, indole.

Alanine↗
PubMed2000

Regulation by external pH and stationary growth phase of the acetolactate synthase from Synechocystis PCC6803.

Several characteristics identify the protein encoded by the alsS gene [sll1981 in Cyanobase (http://www.kazusa.or.jp/cyano/cyano. html)] of Synechocystis PCC6803 as an acetolactate synthase. The AlsS protein is about 60% homologous to the AlsS from Bacillus subtilis or other bacteria. These enzymes condense two pyruvates to form acetolactate, implicated in pH homeostasis via the acetoin-2, 3-butanediol pathway or in valine biosynthesis. Transcriptional fusions revealed that alsS was induced at the onset of stationary phase, as in B. subtilis, a situation leading to an increase in the pHout to above 11 in Synechocystis. This is the first cyanobacterial gene showing a dependence on pH for its expression. Induction was also obtained by the presence of > 100 mM Na+, the effect being prevented by amiloride, in agreement with Na+/H+ exchange in the pH homeostasis process. Homology of the Synechocystis AlsS protein to the close family of acetohydroxy acid synthases (including one in Synechocystis) is around 30%. These enzymes are involved in the parallel routes for valine/leucine and isoleucine biosynthesis. No phenotype of auxotrophy for any of these amino acids was associated with a null mutation in the Synechocystis alsS gene. The AlsS enzyme did not complement the isoleucine deficiency of an acetohydroxy acid synthase-deficient Escherichia coli mutant.

Acetolactate Synthase↗
PubMed1977

Enzyme activities in the sheep placenta during the last three months of pregnancy.

In order to assess the extent to which metabolism within the sheep placenta may influence the transfer of metabolites between mother and foetus at different stages of gestation the activities of enzymes concerned with some aspects of carbohydrate, amino acid and keton body metabolism were determined in placental cotyledons resected from ewes during the last three months of pregnancy. The activities of pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), ATP citrate (pro-3S)-lyase (EC 4.1.3.8), citrate (si)-synthase (EC 4.1.3.7), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA acetyltransferase (EC 2.3.1.9) and 3-keto acid CoA-transferase (EC 2.8.3.5) per gram wet weight cotyledon do not change during the period studied. The activities of alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1), isocitrate dehydrogenase (NADP+) (EC 1.1.1.42), ornithine-oxoacid aminotransferase (EC 2.6.1.13) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) show an increase in activity between the third and fourth months of pregnancy whilst the activities of arginase (EC 3.5.3.1) and possibly pyruvate carboxylase (EC 6.4.1.1) show an increase in activity between the fourth and final months of pregnancy. Ornithine decarboxylase (EC 4.1.1.17) activity declines to one tenth of its activity during this later period. The absence of detectable activities of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) and ornithine carbamoyltransferase (EC 2.1.3.3) indicate that gluconeogenesis and urea synthesis from ammonia do not occur in the sheep placenta. It appears that the ability of the placenta to metabolise several substrates is achieved by the time the placenta reaches its maximum size at approximately 90 days.

Acetoacetates↗
PubMed2002

Diabetes causes marked changes in lymphocyte metabolism.

An enhanced susceptibility to infections is well known to occur in a poorly controlled diabetic state. Since glucose and glutamine are essential for lymphocyte function, we investigated whether their metabolism is changed in lymphocytes obtained from mesenteric lymph nodes of alloxan-induced diabetic rats (40 mg/kg body weight). The activities of hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase (G6PDH), citrate synthase and phosphate-dependent glutaminase were determined. Decarboxylation of metabolites [U-14C]-, [1-14C]- and [6-14C]-glucose, [1-14C]- and [2-14C]-pyruvic acid, [U-14C]-palmitic acid and [U-14C]-glutamine was evaluated in incubated lymphocytes isolated from mesenteric lymph nodes. The measurements were carried out in cells following three experimental protocols: (1) lymphocytes freshly obtained from control and alloxan-induced diabetic rats, (2) lymphocytes from insulin-treated (2 U/rat per day) diabetic rats and (3) lymphocytes obtained from control and diabetic rats and cultured in the presence of insulin (1 mU/ml) for 6 h. The activities of hexokinase, G6PDH and citrate synthase were decreased by the diabetic state, whereas that of phosphofructokinase was raised. Decarboxylation of [U-14C]- and [6-14C]-glucose, [1-14C]- and [2-14C]-pyruvate and [U-14C]-glutamine were also decreased in lymphocytes from diabetic rats, whereas [U-14C]-palmitic acid decarboxylation was increased. Insulin administration in vivo or added to the culture medium reversed the changes observed in freshly obtained lymphocytes. Alloxan-induced diabetes did change lymphocyte metabolism and this may be an important mechanism leading to impairment of lymphocyte function.

Animals↗
PubMed1990

Persistence of gluconeogenesis in Escherichia coli poisoned by oxidant stress.

The poisoning (inhibition of growth rate) of Escherichia coli by 4.2 atmospheres of hyperbaric oxygen was less when the culture energy source was glucose, fructose-6-phosphate, or glycerol, compared to pyruvate, oxaloacetate, or amino acids. This was consistent with previous indirect data which pointed to impaired gluconeogenesis in the toxicity mechanism. However, the three enzymes unique to gluconeogenesis (fructose-1, 6-diphosphatase, phosphoenolpyruvate synthase and phosphoenolpyruvate carboxyl-kinase) were not decreased in specific activity to a biologically significant extent in cell-free extract of cells poisoned by hyperbaric oxygen. Net glycogen synthesis in vivo was not decreased from glycerol, pyruvate or oxaloacetate, compared to glucose in cells exposed to oxidant stress from hyperbaric oxygen or 1 mM aerobic paraquat with cells exposed as exponentially growing cells prior to assay or as resting cells during the assay.

Air Pressure↗
PubMed1995

A double mutant allele, csr1-4, of Arabidopsis thaliana encodes an acetolactate synthase with altered kinetics.

A comparison is made of the kinetic characteristics of acetolactate synthase (EC 4.1.3.18) in extracts from Columbia wild type and four near-isogenic, herbicide-resistant mutants of Arabidopsis thaliana (L.) Heynh. The mutants used were the chlorsulfuron-resistant GH50 (csr1-1), the imazapyr-resistant GH90 (csr1-2), the triazolopyrimidine-resistant Tzp5 (csr1-3) and the multiherbicide-resistant, double mutant GM4.8 (csr1-4), derived from csr1-1 and csr1-2 by intragenic recombination (G. Mourad et al. 1994, Mol. Gen. Genet. 243, 178-184). Kmapp and Vmax values for the substrate pyruvate were unaffected by any of the mutations giving rise to herbicide resistance. Feedback inhibition by L-valine (L-Val), L-leucine (L-Leu) and L-isoleucine (L-Ile) of acetolactate synthase extracted from wild type and mutants fitted a mixed competitive pattern most closely. Ki values for L-Val, L-Leu and L-Ile inhibition were not significantly different from wild type in extracts from csr1-1, csr1-2, and csr1-3. Ki values were significantly higher than wild type by two- and five-fold, respectively, for csr1-4 with L-Val and L-Leu but not L-Ile. GM4.8 (csr1-4) plants were also highly resistant in their growth to added L-Val and L-Leu.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetolactate Synthase↗
PubMed2000

The kinetic mechanism of 5-enolpyruvylshikimate-3-phosphate synthase from a gram-positive pathogen Streptococcus pneumoniae.

The Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a potential novel antibacterial target. The enzyme catalyzes a reversible transfer of an enolpyruvyl group from phospho(enol)pyruvate (PEP) to shikimate 3-phosphate (S3P) to give EPSP with the release of inorganic phosphate (Pi). Understanding the kinetic mechanism of this enzyme is crucial to the design of novel inhibitors of this enzyme that may have potential as antibacterial agents. Steady-state kinetic studies of product inhibition and inhibition by glyphosate (GLP) have demonstrated diverse inhibition patterns of the enzyme. In the forward reaction, GLP is a competitive inhibitor with respect to PEP, but an uncompetitive inhibitor relative to S3P. Product inhibition shows that EPSP is a competitive inhibitor versus both PEP and S3P, suggesting that the forward reaction follows a random sequential mechanism. In the reverse reaction, GLP is an uncompetitive inhibitor versus EPSP, but a noncompetitive inhibitor versus Pi. This indicates that a non-productive quaternary complex might be formed between the enzyme, EPSP, GLP and Pi. Product inhibition in the reverse reaction has also been investigated. The inhibition patterns of the S. pneumoniae EPSP synthase are not entirely consistent with those of EPSP synthases from other species, indicating that EPSP synthases from different organisms may adopt unique mechanisms to catalyze the same reactions.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1977

alpha-Isopropylmalate synthase from Alcaligenes eutrophus H 16. II. Substrate specificity and kinetics.

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following alpha-keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): alpha-ketoisovalerate, alpha-keto-n-valerate, alpha-ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA, malonyl-CoA, valeryl-CoA, and crotonyl0CoA. alpha-Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct L-leucine. The substrate saturation curves of alpha-ketoisovalerate or other alpha-keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between nH-values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (alpha-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 micronM) inactivated the enzyme irreversibly. The 3'-phosphate of coenzyme A and the free carboxyl group of alpha-ketoisovalerate were involved in optimal binding of these substrates, but 3'-dephospho-acetyl-coenzyme A and the methylester of alpha-keto-isovalerate were also converted by this enzyme. A CH3--CH2-grouping of the alpha-keto acids seemed to be necessary for binding this substrate.

2-Isopropylmalate Synthase↗
PubMed1975

Observations on enzymes of ammonia assimilation in two different strains of Cyanidium caldarium.

Two strains of Cyanidium caldarium, one able to utilize nitrate as a substrate, and the other not, were tested for the presence of enzymes of ammonia assimilation. The nitrate-assimilating strain exhibits glutamate dehydrogenase activity. By contrast, the other strain lacks glutamate dehydrogenase; it possesses high alanine dehydrogenase and L-alanine aminotransferase activities which suggest that this strain may incorporate ammonia through reductive amination of pyruvate and may form glutamate from 2-ketoglutarate by a transamination reaction with alanine. Neither strain reveals glutamate synthase activity. Both strains contain similar levels of glutamine synthetase.

Alanine↗
PubMed2006

Identification of developmentally-regulated proteins in Leishmania panamensis by proteome profiling of promastigotes and axenic amastigotes.

We have employed proteomics to identify proteins upregulated in the amastigote life-stage of Leishmaniapanamensis, using axenically-differentiated forms as models of authentic intracellular parasites. Resolution of the soluble proteomes of axenic amastigotes and promastigotes by two-dimensional electrophoresis (2DE) in the neutral pI range (5-7) revealed equivalent numbers of protein spots in both life-stages (644-682 using Coomassie Blue and 851-863 by silver staining). Although representing a relatively low proportion (8.1-10.8%) of the predicted 8000 gene products of Leishmania, these proteome maps enabled the reproducible detection of 75 differentially-regulated protein spots in amastigotes, comprising 24 spots "uniquely" expressed in this life-stage and 51 over-expressed by 1.2-5.7-fold compared to promastigotes. Of the 11 amastigote-specific spots analysed by mass spectrometry (MS), 5 yielded peptide sequences with no orthologues in Leishmania major, and the remaining 6 were identified as 7 distinct proteins (some of which were truncated isoforms) representing several functional classes: carbohydrate/energy metabolism (fructose 1,6-bisphosphate aldolase, glucose 6-phosphate dehydrogenase, pyruvate dehydrogenase), stress response (heat shock protein [HSP] 83), cell membrane/cytoskeleton (beta-tubulin), amino acid metabolism (cysteine synthase) and cell-cycle (ran-binding protein). Four additional over-expressed spots were tentatively identified as HSPs 60 and 70 and HSP 70-related proteins -1 and -4 by positional analogy with these landmark proteins in the Leishmania guyanensis proteome. Our data demonstrate the feasibility of proteomics as an approach to identify novel developmentally-regulated proteins linked to Leishmania differentiation and intracellular survival, while simultaneously pinpointing therapeutic targets. In particular, the amastigote-specific expression of cysteine synthase underlines the importance of de novo cysteine synthesis both as a potential parasite virulence factor and as a major metabolic difference from mammalian host cells.

Amino Acid Sequence↗
PubMed2003

Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry.

Metabolic fluxes provide a detailed metric of the cellular metabolic phenotype. Fluxes are estimated indirectly from available measurements and various methods have been developed for this purpose. Of particular interest are methods making use of stable isotopic tracers as they enable the estimation of fluxes at a high resolution. In this paper, we present data validating the use of mass spectrometry (MS) for the quantification of complex metabolic flux networks. In the context of the lysine biosynthesis flux network of Corynebacterium glutamicum (ATCC 21799) under glucose limitation in continuous culture, operating at 0.1 x h(-1) after the introduction of 50% [1-13C]glucose, we deploy a bioreaction network analysis methodology for flux determination from mass isotopomer measurements of biomass hydrolysates, while thoroughly addressing the issues of measurement accuracy, flux observability and data reconciliation. The analysis enabled the resolution of the involved anaplerotic activity of the microorganism using only one labeled substrate, the determination of the range of most of the exchange fluxes and the validation of the flux estimates through satisfaction of redundancies. Specifically, we determined that phosphoenolpyruvate carboxykinase and synthase do not carry flux at these experimental conditions and identified a high futile cycle between oxaloacetate and pyruvate, indicating a highly active in vivo oxaloacetate decarboxylase. Both results validated previous in vitro activity measurements. The flux estimates obtained passed the chi2 statistical test. This is a very important result considering that prior flux analyses of extensive metabolic networks from isotopic measurements have failed criteria of statistical consistency.

Amino Acids↗
PubMedDate not supplied

Catabolic capacity of the muscles of shorebird chicks: maturation of function in relation to body size.

Newly hatched precocial chicks of arctic shorebirds are able to walk and regulate their body temperatures to a limited extent. Yet, they must also grow rapidly to achieve independence before the end of the short arctic growing season. A rapid growth rate may conflict with development of mature function, and because of the allometric scaling of thermal relationships, this trade-off might be resolved differently in large and small species. We assessed growth (mass) and functional maturity (catabolic enzyme activity) in leg and pectoral muscles of chicks aged 1-16 d and adults of two scolopacid shorebirds, the smaller dunlin (Calidris alpina: neonate mass 8 g, adult mass 50 g) and larger whimbrel (Numenius phaeopus; neonate mass 34 g, adult mass 380 g). Enzyme activity indicates maximum catabolic capacity, which is one aspect of the development of functional maturity of muscle. The growth rate-maturity hypothesis predicts that the development of catabolic capacity should be delayed in faster-growing muscle masses. Leg muscles of both species were a larger proportion of adult size at hatching and grew faster than pectoral muscles. Pectoral muscles grew more rapidly in the dunlin than in the whimbrel, whereas leg muscles grew more rapidly in the whimbrel. In both species and in both leg and pectoral muscles, enzyme activities generally increased with age, suggesting increasing functional maturity. Levels of citrate synthase activity were similar to those reported for other species, but l-3-hydroxyacyl-CoA-dehydrogenase and pyruvate kinase (PK) activities were comparatively high. Catabolic capacities of leg muscles were initially high compared to those of pectoral muscles, but with the exception of glycolytic (PK) capacities, these subsequently increased only modestly or even decreased as chicks grew. The earlier functional maturity of the more rapidly growing leg muscles, as well as the generally higher functional maturity in muscles of the more rapidly growing dunlin chicks, contradicts the growth rate-maturity function trade-off and suggests that birds have considerable latitude to modify this relationship. Whimbrel chicks, apparently, can rely on allometric scaling of power requirements for locomotion and the thermal inertia of their larger mass to reduce demands on their muscles, whereas dunlin chicks require muscles with higher metabolic capacity from an earlier age. Thus, larger and smaller species may adopt different strategies of growth and tissue maturation.

Animals↗
PubMed2005

Breast carcinomas fulfill the Warburg hypothesis and provide metabolic markers of cancer prognosis.

The aim of this study was to investigate selected proteomic markers of the metabolic phenotype of breast carcinomas as prognostic markers of cancer progression. For this purpose, a series of 101 breast carcinomas and 13 uninvolved breast samples were examined for quantitative differences in protein expression of mitochondrial and glycolytic markers. The beta-subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) and heat shock protein 60 (Hsp60), and the glycolytic glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase were identified by immunological techniques. Correlations of the expression level of the protein markers and of the ratios derived from them were established with the clinicopathological information of the tumors and the follow-up data of the patients. The metabolic proteome of breast cancer specimens revealed a pronounced shift towards an enhanced glycolytic phenotype concurrent with a profound alteration on the mitochondrial beta-F1-ATPase/Hsp60 ratio when compared with normal samples. Discriminant analysis using markers of the metabolic signature as predictor variables revealed a classification sensitivity of approximately 97%. Kaplan-Meier survival analysis showed that several of the proteomic variables significantly correlated with overall and disease-free survival of the patients. The expression level of beta-F1-ATPase per se allowed the identification of a subgroup of breast cancer patients with significantly worse prognosis. Multivariate Cox regression analysis indicated that tumor expression of beta-F1-ATPase is a significant marker independent from clinical variables to assess the prognosis of the patients. We conclude that the alteration of the mitochondrial and glycolytic proteomes is a hallmark feature of breast cancer further providing relevant markers to aid in the prognosis of breast cancer patients.

Biomarkers, Tumor↗
PubMed1983

Historical perspectives on protein phosphorylation and a classification system for protein kinases.

Work on the phosphorylation of proteins as a dynamic process involved in the regulation of biological processes started in the 1950s with the finding that phosphorylase a and phosphorylase b are phospho and dephospho forms of the same enzyme. The field expanded sharply in the late 1960s with the discovery of the cyclic-AMP-dependent protein kinase, and it is now clear that phosphorylation-dephosphorylation constitutes a major type of regulation almost as common as allosteric control. The protein kinases, which catalyse the phosphorylation step in various phosphorylation-dephosphorylation systems, can be divided into two main classes, the serine-threonine protein kinases and the tyrosine protein kinases. Each class can be subdivided into groups or entities depending on the nature of the agent(s) that regulate activity.

Animals↗
PubMed1990

Effect of a high-fat-sucrose diet on in vivo insulin receptor kinase activation.

Insulin-stimulated glucose uptake into muscle is depressed by high-fat-sucrose (HFS) feeding of rats. To investigate the mechanism of this insulin resistance, the in vivo activation of the insulin receptor kinase in liver and muscle of control and HFS-fed rats was determined. Rats were injected with glucose and insulin and killed 0, 5, 15, and 30 min after injection. Insulin binding was not changed in partially purified receptors from muscle of HFS rats. In control rats insulin receptor kinase activity was maximally stimulated threefold in liver at 5 min and fourfold in muscle at 15 min after insulin-glucose injection. The insulin-stimulated tyrosine kinase activity of receptors isolated from the liver of rats fed the HFS diet was decreased by 30% in comparison with the controls. In contrast, receptors isolated from muscle did not show any difference in basal or insulin-stimulated kinase activity between HFS-fed and control rats. Decreased in vivo activation of the insulin receptor kinase may be at least partially responsible for insulin resistance in liver. Because insulin binding and insulin stimulation of receptor kinase were normal in muscle of HFS-fed animals, it is concluded that the insulin resistance of glucose uptake into muscle is caused by a defect distal to the insulin receptor.

Animals↗