Formation of transient complexes in the glutamate dehydrogenase catalyzed reaction.

The reaction of glutamate dehydrogenase and glutamate (gl) with NAD+ and NADP+ has been studied with stopped-flow techniques. The enzyme was in all experiments present in excess of the coenzyme. The results indicate that the ternary complex (E-NAD(P)H-kg) is present as an intermediate in the formation of the stable complex (E-NAD(P)H-gl). The identification of the complexes is based on their absorption spectra. The binding of the coenzyme to (E-gl) is the rate-limiting step in the formation of (E-NAD(P)H-kg) while the dissociation of alpha-ketoglutarate (kg) from this complex is the rate-limiting step in the formation of (E-NAD(P)H-gl). The Km for glutamate was 20-25 mM in the first reaction and 3 mM in the formation of the stable complex. The Km values were independent of the coenzyme. The reaction rates with NAD+ were approximately 50% greater than those with NADP+. Furthermore, high glutamate concentration inhibited the formation of (E-NADH-kg) while no substrate inhibition was found with NADP+ as coenzyme. ADP enhanced while GTP reduced the rate of (E-NAD(P)H-gl) formation. The rate of formation of (E-NAD(P)H-kg) was inhibited by ADP, while it increased at high glutamate concentration when small amounts of GTP were added. The results show that the higher activity found with NAD+ compared to NADP+ under steady-state assay conditions do not necessarily involve binding of NAD+ to the ADP activating site of the enzyme. Moreover, the substrate inhibition found at high glutamate concentration under steady-state assay condition is not due to the formation of (E-NAD(P)H-gl) as this complex is formed with Km of 3 mM glutamate, and the substrate inhibition is only significant at 20-30 times this concentration.     

Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.     

Kinetic properties and metabolic contributions of yeast mitochondrial and cytosolic NADP+-specific isocitrate dehydrogenases

To compare kinetic properties of homologous isozymes of NADP+-specific isocitrate dehydrogenase, histidine-tagged forms of yeast mitochondrial (IDP1) and cytosolic (IDP2) enzymes were expressed and purified. The isozymes were found to share similar apparent affinities for cofactors. However, with respect to isocitrate, IDP1 had an apparent Km value approximately 7-fold lower than that of IDP2, whereas, with respect to alpha-ketoglutarate, IDP2 had an apparent Km value approximately 10-fold lower than that of IDP1. Similar Km values for substrates and cofactors in decarboxylation and carboxylation reactions were obtained for IDP2, suggesting a capacity for bidirectional catalysis in vivo. Concentrations of isocitrate and alpha-ketoglutarate measured in extracts from the parental strain were found to be similar with growth on different carbon sources. For mutant strains lacking IDP1, IDP2, and/or the mitochondrial NAD+-specific isocitrate dehydrogenase (IDH), metabolite measurements indicated that major cellular flux is through the IDH-catalyzed reaction in glucose-grown cells and through the IDP2-catalyzed reaction in cells grown with a nonfermentable carbon source (glycerol and lactate). A substantial cellular pool of alpha-ketoglutarate is attributed to IDH function during glucose growth, and to both IDP1 and IDH function during growth on glycerol/lactate. Complementation experiments using a strain lacking IDH demonstrated that overexpression of IDP1 partially compensated for the glutamate auxotrophy associated with loss of IDH. Collectively, these results suggest an ancillary role for IDP1 in cellular glutamate synthesis and a role for IDP2 in equilibrating and maintaining cellular levels of isocitrate and alpha-ketoglutarate.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

Trichomonas vaginalis: characterization of its glutamate dehydrogenase

An NADP-linked glutamate dehydrogenase (EC 1.4.1.4) was found in the soluble fraction of Trichomonas vaginalis. Its molecular weight was about 230,000 (gel filtration). The enzyme, partially purified by diafiltration and hydroxyapatite column chromatography, was heat stable (1 hr at 57 C). It catalyzed both the amination of alpha-ketoglutarate (mean Km 0.6 mM) and the deamination of glutamate (mean Km 1.2 mM) The optimum pH of the amination reaction was 6.7, and that of the deamination reaction was 8. Glutamate was a competitive inhibitor of the amination reaction (mean Ki 5.6 mM) and alpha-ketoglutarate a partially competitive inhibitor of the deamination reaction (mean Ki 0.45 mM). Both guanosine and inosine diphosphates (1 mM) increased the Km alpha-ketoglutarate fivefold (mean Ki's 0.3 and 0.4 mM, respectively). Guanosine diphosphate reduced the Km glutamate 40%. Adenosine di- and triphosphate (1 mM) were ineffective. Because the amination reaction displayed substrate inhibition, guanosine and inosine diphosphates were potent natural inhibitors, and ammonia released by deamination reactions would tend to raise pH (amination operative at acid pH), we hypothesize that the deamination reaction may predominate in the living organism.     

Properties of the thermostable glutamate dehydrogenase of the mesophilic anaerobe Peptostreptoccus asaccharolyticus purified by a novel method after over-expression in an Escherichia coli host

Peptostreptococcus asaccharolyticus glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating); EC 1.4.1.2) overexpressed in Escherichia coli has been purified by two new methods. Enzyme made by the first method showed remarkable thermophilicity, with a temperature optimum of 60 degrees C, and also thermostability, which suggested the second, simpler method, incorporating a heat step. This produced 94 mg of homogeneous protein per litre culture medium. The basic kinetic parameters for P. asaccharolyticus glutamate dehydrogenase with all substrates are revealed at pH 7.0. The enzyme is highly specific for NAD+, with values for kcat/Km 405 times greater than for NADP+. In the reverse direction of reaction, the kcat/Km value for NADH is almost 1000-fold greater than for NADPH.     

Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: evidence for two distinct enzyme forms.

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37 degrees C body temperature) and hibernating (torpid, 5 degrees C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5+/-2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335+/-5 kDa for euthermic and 320+/-5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Km values for glutamate, NH4+, and alpha-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5 degrees C). For example, the Km for alpha-ketoglutarate of euthermic GDH was higher at 5 degrees C (3.66+/-0.34 mM) than at 37 degrees C (0.10+/-0.01 mM), whereas hibernator GDH had a higher affinity for alpha-ketoglutarate at 5 degrees C (Km was 0.98+/-0.08 mM at 37 degrees C and 0.43+/-0.02 mM at 5 degrees C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37 degrees C and weakest with hibernator GDH at 5 degrees C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.     

Partial purification and some properties of delta1-pyrroline-5-carboxylate reductase from Escherichia coli.

delta1-Pyrroline-5-carboxylate (PCA) reductase [L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2] has been purified over 200-fold from Escherichia coli K-12. It has a molecular weight of approximately 320,000. PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not the reverse reaction (even at high substrate concentrations). The partially purified preparation is free of competing pyridine nucleotide oxidase, PCA dehydrogenase, and proline oxidase activities. The Michaelis constant (Km) values for the substrate, PCA, with reduced nicotinamide adenine dinucleotide phosphate (NADPH) or NADH as cofactor are 0.15 and 0.14 mM, respectively. The Km values determined for NADPH and NADH are 0.03 and 0.23 mM, respectively. Although either NADPH or NADH can function as cofactor, the activity observed with NADPH is severalfold greater. PCA reductase is not repressed by growth in the presence of proline, but it is inhibited by the reaction end products, proline and NADP.     

Dirofilaria immitis: comparison of cytosolic and mitochondrial glutamate dehydrogenases

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8-8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and less than 100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of alpha-ketoglutarate reductase from Micrococcus aerogenes

Micrococcus aerogenes grown in media containing glutamate has high levels of glutamate dehydrogenase and alpha-ketoglutarate reductase. The latter enzyme catalyzes the reversible reduction of alpha-ketoglutarate to alpha-hydroxyglutarate in the presence of reduced nicotinamide adenine dinucleotide (NADH). The enzyme has a high specificity for both substrates in either direction and displays Michaelis-Menten kinetics at moderate substrate concentrations. K(m) values of 0.12 to 0.17 mm alpha-ketoglutarate and 0.3 mm NADH for the forward reaction were calculated from data obtained at low substrate concentrations. At high concentrations, this reaction was inhibited by both substrates. The reverse reaction, which proceeded at 0.1 to 0.2 times the rate of the forward reactions, was inhibited by one of the products, alpha-ketoglutarate. K(m) values for the substrates of this reaction were 10 mm for alpha-hydroxyglutarate and 1 mm for nicotinamide adenine dinucleotide. alpha-Ketoglutarate reductase has a molecular weight of 7.5 x 10(4) to 8.2 x 10(4) and is composed of identical polypeptide chains with a molecular weight of 3.6 x 10(4) to 3.8 x 10(4).     

Purification and properties of glutamate synthase from Nocardia mediterranei.

Glutamate synthase was purified about 250-fold from Nocardia mediterranei U32 and characterized. The native enzyme has a molecular weight of 195,000 +/- 5,000 and is composed of two nonidentical subunits with molecular weights of 145,000 +/- 5,000 and 55,000 +/- 3,000. This enzyme is a complex of iron-sulfur flavoproteins with absorption maxima at 278, 375, 410, and 440 nm. It contains 1.1 mol of flavin adenine dinucleotide, 1.0 mol of flavin mononucleotide, 7.5 mol of nonheme iron, and 7.2 mol of acid-labile sulfur per 200,000 g of protein. Km values for L-glutamine, alpha-ketoglutarate, and NADPH were 77, 53, and 110 microM, respectively. The activity of this glutamate synthase is inhibited by its products (i.e., glutamate and NADP), several amino acids, and tricarboxylic acid cycle intermediates.     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

Purification and properties of glutamate synthase from Thiobacillus thioparus.

Glutamate synthase was purified about 250-fold from Thiobacillus thioparus and was characterized. The molecular weight was estimated as 280,000 g/mol. The enzyme showed absorption maxima at 280, 380, and 450 nm and was inhibited by Atebrin, suggesting that T. thioparus glutamate synthase is a flavoprotein. The enzyme activity was also inhibited by iron chelators and thiolbinding agents. The enzyme was specific for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and alpha-ketoglutarate, but L-glutamine was partially replaced by ammonia as the amino donor. The Km values of glutamate synthase for NADPH, alpha-ketoglutarate, and glutamine were 3.0 muM, 50 muM, and 1.1 mM, respectively. The enzyme had a pH optimum between 7.3 and 7.8. Glutamate synthase from T. thioparus was relatively insensitive to feedback inhibition by single amino acids but was sensitive to the combined effects of several amino acids. Enzymes involved in glutamate synthesis in T. thioparus were studied. Glutamine synthetase and glutamate synthase, as well as two glutamate dehydrogenases (NADH and NADPH dependent), were present in this organism. This levels of glutamate synthase and glutamate dehydrogenase were similar in T. thioparus grown on 0.7 or 7.0 mM ammonium sulfate. The sum of the activities of both glutamate dehydrogenases was only 1/25 of that of glutamate synthase under the assay conditions. It was concluded that the glutamine pathway is important for ammonia assimilation in this autotrophic bacterium.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae

Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.     

Purification and properties of the inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana.

The nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) of Chlorella sorokiniana was purified 260-fold to electrophoretic homogeneity in six steps. Depending on the techniques used, the native enzyme appeared to have a molecular weight of 290,000 or 410,000 and to be composed of five to seven identical subunits with a molecular weight of 58,000. The amino acid composition of this enzyme was shown to differ considerably from that of the NAD-GDH in this organism. The NH2-terminal amino acid was unavailable to dansylation. All six cysteines in the native enzyme were in the free sulfhydryl form. The pH optima for the aminating and deaminating reactions were 7.2 and 9.2, respectively. The Km values for NH4+, alpha-ketoglutarate, NADPH, L-glutamate, and NADP+ were 68, 12, 0.13, and 0.038 mM, respectively. At low substrate concentrations, no cooperativity was seen; however, severe inhibition of enzyme activity was observed at high alpha-ketoglutarate concentrations. Nucleotides did not affect enzyme activity. Antiserum produced in rabbits to the subunits of the enzyme yielded a single precipitin band with the purified enzyme in Ouchterlony double-diffusion analysis. Immunoelectrophoresis was used to confirm the purity of the enzyme and also to quantify the amount of enzyme antigen. These studies indicate that the NADPH-GDH and NAD-GDH isozymes are distinct molecular species in this organism.     

Functional evaluation of conserved basic residues in human phosphomevalonate kinase

Phosphomevalonate kinase (PMK) catalyzes the cation-dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate. Loss of basicity can result in catalytically impaired enzymes. On the basis of this precedent, conserved basic residues of human PMK have been mutated, and purified forms of the mutated proteins have been kinetically and biophysically characterized. K48M and R73M mutants exhibit diminished Vmax values in both reaction directions (>1000-fold) with only slight Km perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated Km values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60- and 30-fold, respectively) as well as decreases [50-fold (forward) and 85-fold (reverse)] in Vmax. R84M also exhibits inflated Km values (50- and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The Ki values for R111M and R84M product inhibition by mevalonate 5-diphosphate are inflated by 45- and 63-fold; effects are comparable to the 30- and 38-fold inflations in Km for mevalonate 5-diphosphate. R141M exhibits little perturbation in Vmax [14-fold (forward) and 10-fold (reverse)] but has inflated Km values for ATP and ADP (48- and 136-fold, respectively). The Kd of ATP for R141M, determined by changes in tryptophan fluorescence, is inflated 27-fold compared to wt PMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 and R84 contribute to binding of mevalonate 5-phosphate and R141 to binding of ATP.     

Regulation of Nicotinamide Adenine Dinucleotide Phosphate-specific Glutamate Dehydrogenase in Germinated Spores of Geotrichum candidum

Germinating spores of Geotrichum candidum produce only a nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase. Synthesis of glutamate dehydrogenase was repressed by the presence of ammonia, whereas urea, glutamate, or glutamine were ineffective. The enzyme was not subject to catabolite repression and was localized in the cell sap fraction. The glutamate dehydrogenase has been purified 93-fold and showed maximal activity at pH 8.2 in the forward and reverse directions. When measuring the initial reaction rate at pH 7.2, a variety of tricarboxylic acid cycle intermediates displayed additive and unidirectional activation of the reductive amination reaction and inhibition of the oxidative deamination reaction. The modulating effects were pH-dependent and diminished at alkaline pH values. Substrate inhibition exerted by α-ketoglutarate was strongest at neutral pH.     

Role of phosphate and divalent metal ions in regulation of the activity of the alpha-ketoglutarate dehydrogenase complex from adrenal cortex

Studies of the alpha-ketoglutarate dehydrogenase complex have demonstrated that inorganic phosphate ions cause a decrease in the Km value for alpha-ketoglutarate without changing the maximum reaction rate. In the absence of phosphate (tris-HCl buffer) at low concentrations of alpha-ketoglutarate there are some indications of enzyme-substrate cooperative interactions (the Hill coefficient is 1,6). The cooperativity is removed by ADP, which increases the apparent affinity of the enzyme for alpha-ketoglutarate. Upon divalent cations binding to EDTA in the presence of high (20 mM) concentrations of alpha-ketoglutarate the reaction rate is decreased only by 20%, while the value of Km for the given substrate shows a sharp rise. The nature of Mg2+, Ca2+, Ba2+ and Mn2+ effects on the alpha-ketoglutarate dehydrogenase complex activity depends on their concentration.     

Purification and properties of glutamate dehydrogenase from a thermophilic bacillus.

A 250- to 300-fold purification of a nicotinamide adenine denucleotide phosphate (NADP)-dependent glutamate dehydrogenase (GDH, E.C. 1.4.1.4) with a yield of 60% from a thermophilic bacillus is described. More than one NADP-specific GDH was detected by polyacrylamide gel electrophoresis. The enzyme is of high molecular weight (approximately 2 X 10-6), similar to that of the beef and frog liver GDH. The pI of the thermophilic GDH is at pH 5.24. The enzyme is highly thermostable at the pH range of 5.8 to 9.0. The purified GDH, unlike the crude enzyme, was very labile at subzero temperatures. An unidentified factor(s) from the crude cell-free extract prevented the inactivation of the purified GDH at -70 C. Various reactants of the GDH system and D-glutamate also protected, to some extent, the enzyme from inactivation at -70 C. From the Michaelis constants for glutamate (1.1 X 10-2M), NADP (3 X 10-4M), ammonia (2.1 X 10-2M), alpha-ketoglutarate (1.3 X 10-3M), and reduced NADP (5.3 X 10-5M), it is suggested that the enzyme catalyzes in vivo the formation of glutamate from ammonia and alpha-ketoglutarate. The amination of alpha-ketoglutarate and deamination of glutamate by the thermophilic GDH are optimal at the pH values of 7.2 and 8.4, respectively.     

Ammonium assimilation by Candida albicans and other yeasts: evidence for activity of glutamate synthase

Activities and properties of the ammonium assimilation enzymes NADP+-dependent glutamate dehydrogenase (GDH), glutamate synthase (GOGAT) and glutamine synthetase (GS) were determined in batch and continuous cultures of Candida albicans. NADP+-dependent GDH activity showed allosteric kinetics, with an S0.5 for 2-oxoglutarate of 7.5 mM and an apparent Km for ammonium of 5.0 mM. GOGAT activity was affected by the buffer used for extraction and assay, but in phosphate buffer, kinetics were hyperbolic, yielding Km values for glutamine of 750 microM and for 2-oxoglutarate of 65 microM. The enzymes GOGAT and NADP+-dependent GDH were also assayed in batch cultures of Saccharomyces cerevisiae and three other pathogenic Candida spp.: Candida tropicalis, Candida pseudotropicalis and Candida parapsilosis. Evidence is presented that GS/GOGAT is a major pathway for ammonium assimilation in Candida albicans and that this pathway is also significant in other Candida species.