Essential Metabolic Pathways Research Articles

Human mitochondrial and cytosolic branched-chain aminotransferases are cysteine S-conjugate β-lyases, but turnover leads to inactivation

The mitochondrial and cytosolic branched-chain aminotransferases (BCAT m and BCAT c) are homodimers in the fold type IV class of pyridoxal 5′-phosphate-containing enzymes that also contains d-amino acid aminotransferase and 4-amino-4-deoxychorismate lyase (a β-lyase). Recombinant human BCAT m and BCAT c were shown to have β-lyase activity toward three toxic cysteine S-conjugates [ S-(1,1,2,2-tetrafluoroethyl)- l-cysteine, S-(1,2-dichlorovinyl)- l-cysteine, and S-(2-chloro-1,1,2-trifluoroethyl)- l-cysteine] and toward β-chloro- l-alanine. Human BCAT m is a much more effective β-chloro- l-alanine β-lyase than two aminotransferases (cytosolic and mitochondrial isozymes of aspartate aminotransferase) previously shown to possess this activity. BCAT m, but not BCAT c, also exhibits measurable β-lyase activity toward a relatively bulky cysteine S-conjugate [benzothiazolyl- l-cysteine]. Benzothiazolyl- l-cysteine, however, inhibits the l-leucine–α-ketoglutarate transamination reaction catalyzed by both enzymes. Inhibition was more pronounced with BCAT m. In the presence of β-lyase substrates and α-ketoisocaproate (the α-keto acid analogue of leucine), no transamination could be detected. Therefore, with an amino acid containing a good leaving group in the β position, β-elimination is greatly preferred over transamination. Both BCAT isozymes are rapidly inactivated by the β-lyase substrates. The ratio of turnover to inactivation per monomer in the presence of toxic halogenated cysteine S-conjugates is ∼170–280 for BCAT m and ∼40–50 for BCAT c. Mitochondrial enzymes of energy metabolism are especially vulnerable to thioacylation and inactivation by the reactive fragment released from toxic, halogenated cysteine S-conjugates such as S-(1,1,2,2-tetrafluoroethyl)- l-cysteine. The present results suggest that BCAT isozymes may contribute to the mitochondrial toxicity of these compounds by providing thioacylating fragments, but inactivation of the BCAT isozymes might also block essential metabolic pathways.

Building New Models for Peroxisome Biogenesis

Though many people have never heard of peroxisomes, we cannot survive without them. Peroxisomes are single membrane-bound organelles that participate in a wide variety of essential metabolic pathways in nearly all eukaryotes. These multipurpose organelles contain enzymes for many physiological

Structure and Mechanism of Homoserine Kinase: Prototype for the GHMP Kinase Superfamily

Background: Homoserine kinase (HSK) catalyzes an important step in the threonine biosynthesis pathway. It belongs to a large yet unique class of small metabolite kinases, the GHMP kinase superfamily. Members in the GHMP superfamily participate in several essential metabolic pathways, such as amino acid biosynthesis, galactose metabolism, and the mevalonate pathway. Results: The crystal structure of HSK and its complex with ADP reveal a novel nucleotide binding fold. The N-terminal domain contains an unusual left-handed βαβ unit, while the C-terminal domain has a central α−β plait fold with an insertion of four helices. The phosphate binding loop in HSK is distinct from the classical P loops found in many ATP/GTP binding proteins. The bound ADP molecule adopts a rare syn conformation and is in the opposite orientation from those bound to the P loop–containing proteins. Inspection of the substrate binding cavity indicates several amino acid residues that are likely to be involved in substrate binding and catalysis. Conclusions: The crystal structure of HSK is the first representative in the GHMP superfamily to have determined structure. It provides insight into the structure and nucleotide binding mechanism of not only the HSK family but also a variety of enzymes in the GHMP superfamily. Such enzymes include galactokinases, mevalonate kinases, phosphomevalonate kinases, mevalonate pyrophosphate decarboxylases, and several proteins of yet unknown functions.

Iron regulatory proteins in pathobiology

The capacity of readily exchanging electrons makes iron not only essential for fundamental cell functions, but also a potential catalyst for chemical reactions involving free-radical formation and subsequent oxidative stress and cell damage. Cellular iron levels are therefore carefully regulated in order to maintain an adequate substrate while also minimizing the pool of potentially toxic 'free iron'. Iron homoeostasis is controlled through several genes, an increasing number of which have been found to contain non-coding sequences [i.e. the iron-responsive elements (IREs)] which are recognized at the mRNA level by two cytoplasmic iron-regulatory proteins (IRP-1 and IRP-2). The IRPs belong to the aconitase superfamily. By means of an Fe-S-cluster-dependent switch, IRP-1 can function as an mRNA-binding protein or as an enzyme that converts citrate into isocitrate. Although structurally and functionally similar to IRP-1, IRP-2 does not seem to assemble a cluster nor to possess aconitase activity; moreover, it has a distinct pattern of tissue expression and is modulated by means of proteasome-mediated degradation. In response to fluctuations in the level of the 'labile iron pool', IRPs act as key regulators of cellular iron homoeostasis as a result of the translational control of the expression of a number of iron metabolism-related genes. Conversely, various agents and conditions may affect IRP activity, thereby modulating iron and oxygen radical levels in different pathobiological settings. As the number of mRNAs regulated through IRE-IRP interactions keeps growing, the definition of IRPs as iron-regulatory proteins may in the near future become limiting as their role expands to other essential metabolic pathways.

Biology of Senescent Liver Peroxisomes: Role in Hepatocellular Aging and Disease

Despite rising interest in the health problems of the elderly, information on senescence-related alterations in essential metabolic pathways and their responses to various chemicals is scarce. Although peroxisomal pathways are involved in a multitude of cellular functions, little attention has been given to the potential relationship between senescence of these organelles and the process of aging and disease. Although the prevailing experimental evidence points to a decline in liver peroxisomal enzyme activities and a muted response to peroxisome-proliferating chemicals in aged animals, it is also evident that aged animals are more susceptible, in comparison to their young counterparts, to the hepatocarcinogenic effects of these chemicals. Furthermore, little is known about extraperoxisomal effects of peroxisome proliferators in aged animals. This review evaluates published studies on the impact of aging on basal hepatic peroxisomal metabolism, response to peroxisome proliferators, and changes in signal transduction pathways involved in these processes, with the aim of stimulating research efforts in this important area. The potential intricate relationship among senescent peroxisomes, aged hepatocytes, and health are also discussed.

Biology of senescent liver peroxisomes: role in hepatocellular aging and disease.

Despite rising interest in the health problems of the elderly, information on senescence-related alterations in essential metabolic pathways and their responses to various chemicals is scarce. Although peroxisomal pathways are involved in a multitude of cellular functions, little attention has been given to the potential relationship between senescence of these organelles and the process of aging and disease. Although the prevailing experimental evidence points to a decline in liver peroxisomal enzyme activities and a muted response to peroxisome-proliferating chemicals in aged animals, it is also evident that aged animals are more susceptible, in comparison to their young counterparts, to the hepatocarcinogenic effects of these chemicals. Furthermore, little is known about extraperoxisomal effects of peroxisome proliferators in aged animals. This review evaluates published studies on the impact of aging on basal hepatic peroxisomal metabolism, response to peroxisome proliferators, and changes in signal transduction pathways involved in these processes, with the aim of stimulating research efforts in this important area. The potential intricate relationship among senescent peroxisomes, aged hepatocytes, and health are also discussed.

Mechanism-Based Inhibitors: Development of a High Throughput Coupled Enzyme Assay to Screen for Novel Antimalarials.

Identifying potent enzyme inhibitors through a robust HTS assay is currently thought to be the most efficient way of searching for lead molecules. We have developed a HTS assay that mimics a crucial step in an essential metabolic pathway, the purine salvage pathway of the malarial parasite Plasmodium falciparum. In this assay we have used purified recombinant enzymes: hypoxanthine guanine phosphoribosyl transferase (HGPRT) and inosine monophosphate dehydrogenase (IMPDH) from the malarial parasite and the human host, respectively. These two enzymes, which work in tandem, are used to set up a coupled assay that is robust enough to meet the stringent criteria of an HTS assay. In the first phase of our screen we seem to have identified novel inhibitors that kill the parasite by inhibiting the salvage pathway of the parasite.

Intermediates and products formed during fatty acid alpha-oxidation in cucumber (Cucumis sativus).

Fatty acid alpha-oxidation is an essential metabolic pathway both in plants and in mammals which is still not completely understood. We previously described and purified an alpha-oxidation enzyme in cucumber which has been used in the present investigation of the alpha-oxidation reaction mechanism. Free fatty acids, and not the CoA thioesters, were found to undergo alpha-oxidation in cucumber. 2-Hydroxy- and 2-oxopalmitic acids were identified as palmitic acid alpha-oxidation intermediates by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis in cucumber subcellular 150,000 x g(max) pellets obtained by differential centrifugation. Incubation of purified alpha-oxidation enzyme with [1-14C]palmitic acid resulted in the formation of both the above-described intermediates and the Cn-1 product, pentadecanal, and 14CO2. Besides 14CO2, 14C-formate was identified as an alpha-oxidation product from [1-14C]palmitic acid in cucumber subcellular fractions. Fe2+ stimulated the 14CO2 and 14C-formate production, and the addition of ascorbate and 2-oxoglutarate together with Fe2+ resulted in optimal alpha-oxidation activities, suggesting a dioxygenase reaction mechanism, as previously shown in mammals. NADPH and, to a lesser extent, NADH stimulated the total 14C-formate plus 14CO2 production but had only slight or no effects on 14CO2 production. H2O2 showed concentration-dependent inhibitory effects, while FAD had neither effect on 14CO2 nor 14CO2 plus 14C-formate production. The results in the present study demonstrate that an alpha-oxidation enzyme in cucumber is capable of oxidizing palmitic acid via 2-hydroxy- and 2-oxo-palmitic acid to produce pentadecanal and CO2. In contrast to the subcellular 150,000 x g(max) fraction, the purified alpha-oxidation enzyme could neither produce formate nor convert 14C-formate into 14C02, indicating two possible alpha-oxidation routes in cucumber.

‘Spectrinase’ activity

Malaria claims 1.7–2.5 million lives each year and is caused by Plasmodium, an intra-erythrocytic protozoan that is transmitted by the bite of an infected female Anopheles mosquito. Efforts to combat the disease have been thwarted by Plasmodium strains resistant to anti-malarial drugs and by mosquitoes resistant to insecticides. New anti-malarial strategies are focusing on blocking the essential metabolic pathways of the parasite.

Plasma pyridoxal phosphate and pyridoxic acid and their relationship to plasma homocysteine in a representative sample of British men and women aged 65 years and over

Concentrations of pyridoxal phosphate and pyridoxic acid were measured in fasting plasma samples from British men and women aged 65 years and over, participating in a National Diet and Nutrition Survey during 1994-5, selected to be representative of the population of mainland Britain. In this population, the concentration of pyridoxal phosphate declined, whereas pyridoxic acid rose, with increasing age and frailty; however, both status indicators were strongly and directly (with a positive coefficient) correlated with estimates of vitamin B6 intake. This was little affected by the inclusion of food energy and protein intakes in the model. Forty-eight percent of the participants living in the community and 75% of those living in institutions had plasma pyridoxal phosphate concentrations below a range considered normal from other studies. In a univariate regression model, plasma pyridoxal phosphate concentrations were inversely correlated with plasma homocysteine concentrations, consistent with the hypothesis that vitamin B6 status may influence plasma homocysteine levels, and hence vascular disease risk. However, this relationship was partly attenuated in a multiple regression model including age, sex, domicile and biochemical status indices, including those of folate and vitamin B12. There was evidence that plasma pyridoxal phosphate was sensitive to metabolic conditions associated with inflammation and the acute-phase reaction, and that plasma pyridoxic acid was sensitive to renal function. Thus, neither index is an ideal predictor of vitamin B6 status in older people, unless these confounding factors are allowed for. Since poor vitamin B6 status may have health implications, e.g. for immune function, cognition, and for essential intermediary metabolic pathways in older people, it needs to be investigated as a possible public health problem.

Carnosine: an endogenous neuroprotector in the ischemic brain.

1. The biological effects of carnosine, a natural hydrophilic neuropeptide, on the reactive oxygen species (ROS) pathological generation are reviewed. 2. We describe direct antioxidant action observed in the in vitro experiments. 3. Carnosine was found to effect metabolism indirectly. These effects are reflected in ROS turnover regulation and lipid peroxidation (LPO) processes. 4. During brain ischemia carnosine acts as a neuroprotector, contributing to better cerebral blood flow restoration, electroencephalography (EEG) normalization, decreased lactate accumulation, and enzymatic protection against ROS. 5. The data presented demonstrate that carnosine is a specific regulator of essential metabolic pathways in neurons supporting brain homeostasis under unfavorable conditions.

The minimal cell genome: "on being the right size".

The minimal cell genome: "on being the right size".

Gene expression on acute myocardial stress. Induction by hypoxia, ischemia, reperfusion, hyperthermia and oxidative stress

It is apparent from the above discussion that acute stress, such as ischemia and reperfusion, hypoxia and reoxygenation, hyperthermia and oxidative stress, can rapidly potentiate the induction of genes for certain members of the HSP families and for antioxidants/antioxidant enzymes. Whether the stress response and induction of these genes have a direct role in myocardial protection is not known, but the induction of the expression of these genes are mostly associated with the preservation of myocardial cells from subsequent injury resulting from ischemia, hypoxia and reperfusion. The ubiquitous presence of some of these stress genes, such as for HSP 70 and catalase, in normal unstressed myocardium further suggests a role of these genes in many basic and essential biochemical and metabolic pathways. It is reasonable to speculate that the cells respond to the stress as a consequence of perturbations of one or more of the metabolic pathways by stimulating the induction of the stress genes of that particular pathway in which they participate. Thus, these genes are likely to be involved both in the protection and recovery/repair mechanisms. The precise mechanism by which myocardial cell recognizes and responds to a particular stress agent such as ischemia, hypoxia, hyperthermia or oxidative stress is not clear. While it is tempting to speculate that a generalized mechanism exists, applying to all different modes of stress response and gene induction, whether these agents induce the response via independent pathways or converge within a single point is entirely unclear. However, from the striking resemblance between the pattern of gene expression, especially with regard to HSP and antioxidant genes, it is reasonable to hypothesize the existence of a common and essential pathway of molecular signaling that leads to the expression of these stress genes (Fig. 2). The identification and characterization of the transcription factors that regulate the expression of the genes induced by these forms of stress should greatly facilitate our future understanding of the mechanism of stress response.

Mechanism-based inhibition of proline dehydrogenase by proline analogues

The inactivation of proline dehydrogenase by several l-Pro analogues was investigated with the aim to block the essential metabolic pathway of tsetse flies allowing the degradation of l-Pro to l-Glu. In vitro studies on rat liver mitochondria showed that only 4-methylene- l-proline was able to inactivate proline dehydrogenase. The inactivation kinetics agreed with a mechanism-based inhibition. The other tested analogues E- and Z-4- fluoromethylene- l-proline , and cis and trans-5- ethyl- d,l-proline were neither substrate nor inactivator of the enzyme. In vivo 4-methylene- l-proline showed no toxicity against Drosophila flies, but was lethal for Glossina pallidipes flies. This result allows the consideration of 4-methylene- l-proline as an attractive compound molecule in the struggle against tsetse flies.

Phosphate accumulation and the occurrence of polyphosphates and cyclic 2,3-diphosphoglycerate in Methanosarcina frisia

Methanosarcina frisia accumulates phosphate up to 14% of its dry weight. The phosphate is stored as long-chain polyphosphates as shown by 31P-NMR investigations. Further results show that the accumulation of phosphates is substrate-dependent. In the presence of H2 and CO2 as the only carbon and energy source 180 mg of PO inf4 sup3- /g protein were accumulated, whereas 260 mg PO inf4 sup3- /g protein were accumulated in the presence of methanol. This is far more than necessary for the maintenance of essential metabolic pathways. In addition, the 31P-NMR studies show the occurrence of cyclic 2,3-diphosphoglycerate in Methanosarcina frisia. The role of the phosphate metabolites in cell metabolism are discussed.

Metabolic Control of Luminescence in the Luminous Organs of the Teleost Porichthys: Effects of the Metabolic Inhibitors Iodoacetic Acid and Potassium Cyanide

ABSTRACT The treatment of isolated photophores of the epipelagic luminescent fish Porichthys notatus Girard with the glycolytic inhibitor iodoacetic acid (IAA) induces light production. This luminescence is not significantly affected by the adrenergic antagonists phentolamine and propranolol. The addition of pyruvate, phosphoenolpyruvate or glyceraldehyde 3-phos-phate in saline suppresses the light response to IAA. D-Glucose, D-glyceraldehyde, and 2-and 3-phosphoglycerate have no inhibitory effects. The luminescence of the photophores induced by KCN is inhibited by D-glucose, D-glyceraldehyde 3-phosphate and 3-phosphoglycerate. In contrast, 2-deoxy-D-glucose, pyruvate, phosphoenolpyruvate, D-glyceraldehyde and 2-phosphoglycerate do not affect this light production. These results confirm the view that glycolysis and oxidative phosphorylation are essential metabolic pathways in the photogenic cells. They further indicate that common products of these tightly associated metabolic pathways may regulate the activity of the luminous system in these cells.

The role of unnatural dietary trans and cis unsaturated fatty acids in the epidemiology of coronary artery disease

During 1956 the first report on the hypocholesterolemic effect of unsaturated fatty acids of plant and marine origin was published in The Lancet. Consequently it was stated in a Lancet Editorial that hydrogenation of vegetable oils could have contributed to the causation of coronary artery disease and predicted that a decade would probably be required to resolve this question. However, after the lapse of three decades this issue would appear to be no nearer to a clear answer now than it was then. During 1956 hydrogenation was assumed to effect only a reduction in the level of unsaturated fatty acids in the products produced from hydrogenated oils. However, since that time essential fatty acid metabolic pathways to eicosanoids were discovered and described. Also large quantities of unnatural trans and cis unsaturated fatty acids have been shown to form during hydrogenation and these occur in margarines and many other common foods in high concentrations. It has also been shown that these unnatural trans and cis fatty acids block essential fatty acid metabolism by the competitive inhibition of the desaturase enzyme Δ-6-desaturase. Therefore some of the possible metabolic mechanisms whereby “hydrogenation plants could have contributed to the causation of a major disease” have become clearer during the last three decades. Despite a recent conclusion by an ad hoc FDA panel that there need be little concern about the effects of trans fatty acids in the American diet on health, it is nevertheless proposed that on the basis of available evidence, unnatural dietary trans and cis unsaturated fatty acid isomers should be regarded as a definite risk factor in the etiology of coronary artery disease.

Biochemical basis for improving chemotherapeutic regimens in liver malignancies.

As long as it is impossible to prevent or to correct the altered expression of genetic information in malignant cells, tumor chemotherapy has to aim at the elimination of malignant cells by means of cytostatic or cytocidal drugs that are as selective as possible for a given tumor. The minimal difference in the sensitivity of normal and neoplastic cells remains a critical limitation in the use of effective agents, many of which are antimetabolites that inhibit enzymes of essential metabolic pathways on the basis of a structural similarity with physiological intermediates, interfering thereby with cellular growth and proliferation. This may be illustrated by the blockade of thymidylate synthetase induced by 5-fluoro-5′-deoxyuridinemonophosphate (FdUMP), a metabolite of the pyrimidine analog 5-fluorouracil (reviewed by Heidelberger [5]. The consequent depletion of deoxythymidine-5′-monophosphate (dTMP) and deoxythymidine-5′-triphosphate (dTTP) pools should result in DNA synthesis inhibition due to substrate deficiency. This is one of several mechanisms by which fluorinated pyrimidines preferentially inhibit proliferating cells. This effect may be circumvented, however, by dTMP formation from deoxythymidine on the salvage pathway, and this underlines the critical importance of quantitative metaboliteantimetabolite relationships in tumor chemotherapy.

Diverse forms of stress lead to new patterns of gene expression through a common and essential metabolic pathway.

Many eukaryotic organisms respond to heat shock by synthesizing new proteins. We examined the possibility that heat shock proteins represent a particular expression of a general response to stress and that, regardless of the nature of the effective stimulus, the same proteins are synthesized. Accordingly, cardiac stress was applied in the intact rat by four methods: banding the ascending aorta, increasing body temperature to 42 degrees C, reducing body temperature to 18 degrees C, and forcing the rat to swim until exhausted. The hearts were then extirpated and analyzed for new mRNA synthesis. The extracted RNA was translated in a cell-free medium containing [35S]methionine. Translation products were resolved by two-dimensional electrophoresis and visualized by autoradiography. Lactic acid concentration in heart tissue was determined enzymatically. The results showed that two new and distinct proteins of Mr 71,000 and isoelectric points of 5.8 and 6.1 were synthesized in hearts stressed by banding and by heating but not in hearts of exhausted swimmers or in animals at reduced body temperatures. There was no significant difference in cardiac lactic acid concentration between control hearts and hearts from swimmers or cold-treated animals. However, there was a 2-fold increase in lactic acid concentration in hearts of rats with banded aortas compared to controls and a 10-fold increase in heat shocked hearts. We conclude that, under conditions in which the energy requirements of the heart are not completely met by aerobic processes, the resultant lactic acidosis creates an intracellular environment that leads to the selective activation of genes, the production of new mRNA, and the synthesis of a typical group of stress proteins.

High pressure effects on the activity of glycolytic enzymes.

High hydrostatic pressure inhibits growth in most organisms; this may be explained by a deactivation of enzymes involved in essential metabolic pathways. In order to check this hypothesis the enzymic activity of rabbit muscle lactic dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase was investigated in the presence of the coenzyme and excess of substrate at pressures up to 2kbar. Kinetic analysis of an initial phase of pressure induced activation and of a second phase of reversible deactivation shows that the two enzymes respond to high pressures in different ways leading to a volume of activation of increment V is not equal to (LDH) equal 0 plus or minus 1 cm-3 mol-1 and increment V is not equal to (GAPDH) equals 60 plus or minus 4 cm-3 mol-1, respectively. Comparing the lower limits of pressure deactivation, LDH is found to be more stable towards pressure than GAPDH. At p is approximately equal to 2 kbar total deactivation of both enzymes is observed. A concentration dependent lag of GAPDH reactivation proves dissocation to participate in the process of deactivation, while the effects for LDH are explicable on the basis of reversible denaturation alone.