Forms Of Glutamate Dehydrogenase Research Articles

Effect of Chemical Chaperones on the Stability of Proteins during Heat- or Freeze-Thaw Stress.

The importance of studying the structural stability of proteins is determined by the structure-function relationship. Protein stability is influenced by many factors among which are freeze-thaw and thermal stresses. The effect of trehalose, betaine, sorbitol and 2-hydroxypropyl-β-cyclodextrin (HPCD) on the stability and aggregation of bovine liver glutamate dehydrogenase (GDH) upon heating at 50 °C or freeze-thawing was studied by dynamic light scattering, differential scanning calorimetry, analytical ultracentrifugation and circular dichroism spectroscopy. A freeze-thaw cycle resulted in the complete loss of the secondary and tertiary structure, and aggregation of GDH. All the cosolutes suppressed freeze-thaw- and heat-induced aggregation of GDH and increased the protein thermal stability. The effective concentrations of the cosolutes during freeze-thawing were lower than during heating. Sorbitol exhibited the highest anti-aggregation activity under freeze-thaw stress, whereas the most effective agents stabilizing the tertiary structure of GDH were HPCD and betaine. HPCD and trehalose were the most effective agents suppressing GDH thermal aggregation. All the chemical chaperones stabilized various soluble oligomeric forms of GDH against both types of stress. The data on GDH were compared with the effects of the same cosolutes on glycogen phosphorylase b during thermal and freeze-thaw-induced aggregation. This research can find further application in biotechnology and pharmaceutics.

Glutamate Dehydrogenase: Structure, Allosteric Regulation, and Role in Insulin Homeostasis

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine and inhibitors include GTP, palmitoyl CoA, and ATP. Spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds blocked the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

Intertissue Differences for the Role of Glutamate Dehydrogenase in Metabolism

The enzyme glutamate dehydrogenase (GDH) plays an important role in integrating mitochondrial metabolism of amino acids and ammonia. Glutamate may function as a respiratory substrate in the oxidative deamination direction of GDH, which also yields α-ketoglutarate. In the reductive amination direction GDH produces glutamate, which can then be used for other cellular needs such as amino acid synthesis via transamination. The production or removal of ammonia by GDH is also an important consequence of flux through this enzyme. However, the abundance and role of GDH in cellular metabolism varies by tissue. Here we discuss the different roles the house-keeping form of GDH has in major organs of the body and how GDH may be important to regulating aspects of intermediary metabolism. The near-equilibrium poise of GDH in liver and controversy over cofactor specificity and regulation is discussed, as well as, the role of GDH in regulation of renal ammoniagenesis, and the possible importance of GDH activity in the release of nitrogen carriers by the small intestine.

Insights into theIn VivoRegulation of Glutamate Dehydrogenase from the Foot Muscle of an Estivating Land Snail

Land snails, Otala lactea, survive in seasonally hot and dry environments by entering a state of aerobic torpor called estivation. During estivation, snails must prevent excessive dehydration and reorganize metabolic fuel use so as to endure prolonged periods without food. Glutamate dehydrogenase (GDH) was hypothesized to play a key role during estivation as it shuttles amino acid carbon skeletons into the Krebs cycle for energy production and is very important to urea biosynthesis (a key molecule used for water retention). Analysis of purified foot muscle GDH from control and estivating conditions revealed that estivated GDH was approximately 3-fold more active in catalyzing glutamate deamination as compared to control. This kinetic difference appears to be regulated by reversible protein phosphorylation, as indicated by ProQ Diamond phosphoprotein staining and incubations that stimulate endogenous protein kinases and phosphatases. The increased activity of the high-phosphate form of GDH seen in the estivating land snail foot muscle correlates well with the increased use of amino acids for energy and increased synthesis of urea for water retention during prolonged estivation.

Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

Regulation of liver glutamate dehydrogenase by reversible phosphorylation in a hibernating mammal

Glutamate dehydrogenase (GDH) is a key enzyme that links amino acid and carbohydrate metabolism in cells. Regulation is likely most important when organisms are confronted with extreme stresses such as the low environmental temperatures and lack of food associated with winter. Many small mammals, such as Richardson's ground squirrels, Spermophilus richardsonii, cope with these conditions by hibernating. Animals enter long periods of profound torpor where metabolic rate is greatly suppressed, body temperature drops to near-ambient and all metabolic needs must be met from fixed internal body stores of fuels. To investigate how GDH is regulated under these conditions, kinetic properties of GDH were analyzed in liver from euthermic and torpid squirrels, revealing significant differences in V(max), K(m) glutamate, K(a) ADP and inhibition by urea between the two forms of GDH. These data suggested an activation of the glutamate-oxidizing activity of GDH in the hypometabolic state. Subsequent experiments suggested that the molecular basis of the kinetic differences was a change in the protein phosphorylation state of GDH between euthermia and torpor. Specifically, liver GDH appears to be dephosphorylated and activated when animals transition into torpor and this may serve to promote amino acid oxidation to contribute to energy production and gluconeogenesis. This is the first study to show that mammalian liver GDH can be regulated by reversible phosphorylation, providing an important new regulatory mechanism for GDH control.

Bypassing Isophthalate Inhibition by Modulating Glutamate Dehydrogenase (GDH): Purification and Kinetic Characterization of NADP-GDHs from Isophthalate-Degrading Pseudomonas aeruginosa Strain PP4 and Acinetobacter lwoffii Strain ISP4

Pseudomonas aeruginosa strain PP4 and Acinetobacter lwoffii strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; GDH(I) was produced when cells were grown on isophthalate, while GDH(II) was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, GDH(P), when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. GDH(I) and GDH(II) were found to be homotetramers, while GDH(P) was found to be a homohexamer. GDH(II) was more sensitive to inhibition by isophthalate (2.5- and 5.5-fold more sensitive for amination and deamination reactions, respectively) than GDH(I). Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, GDH(I) and GDH(II), in strain PP4. In strain ISP4, irrespective of the carbon source, the GDH(P) produced showed similar levels of inhibition with isophthalate. However, the specific activity of GDH(P) from isophthalate-grown cells was 2.5- to 3-fold higher than that of GDH(P) from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of GDH(P) in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy.

Novel Inhibitors Complexed with Glutamate Dehydrogenase: ALLOSTERIC REGULATION BY CONTROL OF PROTEIN DYNAMICS

Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)(+) as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

Diversity of glutamate dehydrogenase in human brain

Three forms of glutamate dehydrogenase (GDH, EC 1.4.1.3) are purified from human brain tissue. Two of them, named GDH I (consisting of 58±1-kDa subunit) and GDH II (consisting of 56±1-kDa subunit), are readily solubilized and the third one, GDH III (consisting of 56±1-kDa subunit), is a membrane-associated (particulate bound) isoform. Kinetic constants were determined for GDH III. These GDH forms were found to differ in hydrophobicity as indicated by different affinity to Phenyl-Sepharose. All three GDH forms showed microheterogeneity on two-dimensional (2-D) gel electrophoresis. Specific polyclonal antibodies, which enable to determine the levels of immunoreactivities of all the GDH forms in human brain extracts by enzyme-chemiluminescent amplified (ECL)-Western immunoblotting, were obtained.

Isoenzymes from Hulls and Seeds of Developing Pea Fruits

We have surveyed the distribution of multiple forms of thirteen enzyme activities in the pod wall (hull), seedcoats, cotyledons and embryonic axes from developing pods of the garden pea ( Pisum sativum L.). Many profiles reveal forms that appear to be confined to specific organs. Three forms of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) are confined to the embryonic axis; one form of phosphoglucoisomerase (EC 5.3.9.1) and two forms of glutamate dehydrogenase (EC 1.4.1.3) are confined to the embryo; one form of phosphoglucomutase (EC 2.7.5.1) and two forms of malate dehydrogenase (EC 1.1.1.37) are confined to the hull. For most activities, there are more isoenzymes in the reproductive organs than have hitherto been identified from leaves. This information should stimulate further studies on the properties, subcellular locations and functions of multiple enzyme forms, and assist the development of genetic linkage maps, which have so far depended substantially on leaf isoenzymes.

Denaturation of bovine liver glutamate dehydrogenase by guanidine hydrochloride. Correlation between enzymatic activity and molecular state.

Bovine liver glutamate dehydrogenase (GDH), a hexameric enzyme, undergoes subunit dissociation, denaturation, and inactivation in the presence of guanidine hydrochloride (GdnHCl), depending on the denaturant concentration. The correlation between the enzymatic activity and the molecular state of GDH, and the reconstitution of native hexamer from subunits after the removal of GdnHCl were examined by measuring the enzymatic activity and CD spectrum in the far ultraviolet region. It was found that only the hexameric form of GDH has enzymatic activity, and the reconstitution of the hexamer with full enzymatic activity from the trimeric form which has native polypeptide chain structure can be achieved by the removal of GdnHCl. On the other hand, the recovery of enzymatic activity from the dissociated form in more concentrated GdnHCl solution where unfolding of the polypeptide chain takes place showed an exponential decrease with increasing incubation time in the GdnHCl solution. The time constant for the decay of enzymatic activity with respect to the incubation time was almost the same as that for unfolding of the polypeptide chain (followed by CD spectroscopy). It is suggested on the basis of these experimental results that the failure of reconstitution of GDH hexamer from subunits produced at high denaturant concentration is due to failure in the refolding of the unfolded subunit to the correct three-dimensional structure of the polypeptide chain rather than in the reassociation process from subunits.

The chymotrypsin-catalysed activation of bovine liver glutamate dehydrogenase.

1. Ox liver glutamate dehydrogenase is activated by bovine pancreatic alpha-chymotrypsin, but the extent of activation is dependent on the age of the dehydrogenase preparation. 2. The degree of activation is constant and the pseudo-first-order rate constant of activation is directly proportional to the concentration of proteinase used. 3. Commercial preparations of alpha-chymotrypsin differ in their ability to produce a secondary inactivation phase, and this was shown to be due to low tryptic contamination. The 'superactive' form of glutamate dehydrogenase has an increased sensitivity to tryptic inactivation as compared with the native enzyme. 4. Analysis of the activation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the subunit molecular weight of 'superactive' glutamate dehydrogenase differs by less than 5% from that of the native subunit.

Glutamate dehydrogenase activity in developing soybean seed: Kinetic properties of three forms of the enzyme

The kinetic parameters of three forms of glutamate dehydrogenase from developing soybean seed have been determined. For all enzymes double reciprocal plots were intersecting in animating and deaminating directions when each substrate was held at a fixed concentration in turn. In amination all plots were linear and the K NH4 + and K NAD(P)H values determined using (NH 4) 2SO 4 were about one-fifth of values obtained with NH 4CL. In deamination, plots were linear when l-glutamate and NADP + were varied and biphasic with varied NAD +. It was not possible to determine the mechanisms of the enzymes but the data are consistent with nucleotide binding to the free enzyme forms. The role of the enzymes in the tissue is discussed. They could be active in deamination in the later stages of seed development when the supply of carbohydrate could be limiting. They could also be important in ammonia assimilation and their kinetic properties are discussed in relation to known properties of glutamine synthetase and glutamate synthase and cellular levels of enzyme substrates.

Glutamate dehydrogenase activity in developing soybean seed: Isolation and characterization of three forms of the enzyme

Three forms of glutamate dehydrogenase have been isolated from developing soybean seed. Their intracellular locations could not be determined directly because organelles and marker enzymes showed abnormal distribution on sucrose density gradient fractionation. By analogy with enzymes from other parts of the plant, glutamate dehydrogenase 2 was shown to be located in chloroplasts and glutamate dehydrogenase 3 in mitochondria. Glutamate dehydrogenase 1 could not be located in this way because it is found only in the seed. The three enzymes are similar in pH optima, molecular weight and substrate specificity with respect to 2-oxoglutarate and l-glutamate. The mitochondrial enzyme is specific for NAD +. The chloroplast enzyme shows low activity with NADP + relative to NAD + but uses NADPH readily in the aminating direction. Glutamate dehydrogenase 1 is active with both nucleotides and is the only form to show substantial deaminating activity with NADP +. Glutamate dehydrogenases 1 and 2 are activated and stabilized by glutathione and 2-mercaptoethanol whereas enzyme 3 is unaffected. No significant metabolic control of any of the enzymes could be detected. Malate, citrate, adenine nucleotides and long-chain fatty acyl CoA derivatives gave slight inhibition at high concentrations. Amino acids had no effect on activity. A possible role for the enzyme characteristic of the developing seed is discussed in relation to nutrient supply during the accumulation of reserve materials in the seed.

The Enzyme-Enzyme Complex of Transaminase and Glutamate Dehydrogenase

Glutamate dehydrogenase decreases the distribution coefficient of glutamate oxalacetate transaminase in Sephadex G-200. This is consistent with previous results which suggested that a complex is formed between these two enzymes. These gel filtration as well as kinetic experiments suggest that transaminase can react with monomeric but not polymer forms of glutamate dehydrogenase. When the levels of both mitochondrial enzymes are too low to form a complex, there is little TPNH and NH4+ generated by the combined aspartate aminotransferase and glutamate dehydrogenase reactions. This is because oxalacetate is a potent product inhibitor of transaminase, the reaction with glutamate and glutamate dehydrogenase is slow and α-ketoglutarate, oxalacetate, and aspartate all inhibit this latter reactions. When the levels of both enzymes are sufficiently high to form a complex, asparate can be dehydrogenated quite rapidly even in the absence of α-ketoglutarate. Furthermore, the aspartate dehydrogenase reaction catalyzed by the enzyme-enzyme complex is not markedly inhibited by oxalacetate, or activated by α-ketoglutarate, and can take place even in the presence of rather high levels of glutamate. Kinetic and gel filtration experiments suggest that the dissociation constant of the enzyme-enzyme complex is considerably lower than that of these substrates for the free enzymes. These results suggest that an important physiological function of the enzyme-enzyme complex is to catalyze the asparatate dehydrogenase reaction in organs as brain, liver, and kidney, where the mitochondrial levels of these enzymes are sufficiently high to form a complex. An advantage of catalysis by the complex over transamination with aspartate followed by dehydrogenation of glutamate is that the complex is not markedly inhibited by low levels of oxalacetate. When tyrosine is the substrate and the levels of these two enzymes are too low to form a significant amount of complex, the tyrosine aminotransferase and glutamate dehydrogenase reactions are coupled. This can occur with tyrosine but not aspartate because hydroxyphenylpyruvate is not a potent inhibitor. However, when the levels of both enzymes are sufficiently high to form a significant amount of the enzymeenzyme complex, the tyrosine dehydrogenase reaction can be catalyzed by this complex and again α-ketoglutarate has no effect on the reaction. Similar relationships occur when phenylalanine is the substrate. Thus, the enzyme-enzyme complex can facilitate dehydrogenation of these amino acids which do not react with glutamate dehydrogenase.

NAD and NADP-dependent glutamate dehydrogenase in Hydrogenomonas H 16.

Hydrogenomonas H 16 synthetized two chromatographically distinct forms of glutamate dehydrogenase which differed in their thermolability. One glutamate dehydrogenase utilized NAD, the other NADP as a coenzyme.

COMPLEMENTATION RELATIONSHIP OF NEUROSPORA AM MUTANTS IN RELATION TO THEIR FORMATION OF ABNORMAL VARIETIES OF GLUTAMATE DEHYDROGENASE.

Six newammutants ofNeurospora,am14–am19, with defective formation of NADP-linked glutamate dehydrogenase, were isolated following treatment of conidia with nitrous acid. This brings the number of genetically distinct and viableammutants to sixteen. Heterocaryon-compatible isolates of each of the new mutants were tested for complementation with each of the mutants previously known. A number of new complementary combinations were found which add to the complexity of the complementation map which, however, remains linear.The mutants which show complementation aream1,am2,am3,am7,am14andam19. It has proved possible to isolate an altered form of glutamate dehydrogenase from each of these exceptam14, which appears to produce no protein fractionating like the normal enzyme. Among the non-complementing mutants,am4produces an easily-isolated inactive form of glutamate dehydrogenase. These results agree with the immunochemical findings of Roberts & Pateman (1964).Of the mutationally altered forms of the enzyme, theam1,am2,am3andam7varieties are electrophoretically identical or closely similar to the normal protein, while theam4andam19varieties move faster towards the anode at pH 8·5,am19being the faster of the two. Theam19protein, like theam2andam3proteins previously reported on, can be activated to give some enzyme activity, but the activity obtained is much lower for this mutant than foram2oram3.All the complementary pairs of mutants, including those involvingam19, form active enzyme varieties which have very close to normal electrophoretic properties. In several cases the complementation enzyme is less stable to heat than the wild-type enzyme.

Different structural forms of reversibly dissociated glutamic dehydrogenase: Relation between enzymatic activity and molecular weight

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.