NAD kinase interaction with the activator--glutamate dehydrogenase

An electrophoretically homogeneous preparation of the NAD kinase activating factor was isolated from rabbit liver and its physico-chemical properties were investigated. The similarity of molecular weights of the activator subunit and hexamer, pI values, the number of SH-groups to the corresponding parameters for glutamate dehydrogenase and the glutamate dehydrogenase activity demonstrated by this factor allowed for the identification of the NAD kinase activating factor as glutamate dehydrogenase. Using three independent methods, the formation of the NAD kinase--glutamate dehydrogenase complex was shown. Both the oligomeric and monomeric (subunit) forms of NAD kinase were found to be able to form complexes with glutamate dehydrogenase.     

Study of the complex formation between alpha- and beta- forms of NAD-kinase and glutamate dehydrogenase and the effect of metabolites on enzyme interaction

The alpha- and beta-forms of rabbit liver NAD kinase were found to differ significantly in terms of their ability to form complexes with glutamate dehydrogenase. The beta-form of the enzyme was shown to form a more stable complex with glutamate dehydrogenase (Kd = 1.5.10(-8) M), whereas the Kd value for the alpha-form is 2.9.10(-7) M. Using two independent methods, it was shown that in the absence of effectors 40% of the beta-form of NAD kinase and up to 20% of the alpha-form are bound to glutamate dehydrogenase. The substrates of NAD kinase markedly activate the complex formation only in the case of the alpha-form of the enzyme. The time needed for this process is also reduced in the presence of the substrates.     

Reactivation of the phospho form of the NAD-dependent glutamate dehydrogenase by a yeast protein phosphatase

A protein phosphatase was isolated from the yeast, Candida utilis, which could reactivate (dephosphorylate) the phosphorylated form of the NAD-dependent glutamate dehydrogenase. The protein could also dephosphorylate casein, histone and kemptide (a heptapeptide corresponding to the phosphorylation site of liver pyruvate kinase). Reactivation of the phosphorylated glutamate dehydrogenase was stimulated by the simultaneous addition of NAD and L-glutamate; 2-oxoglutarate, NH+4 and NADH had no effect. The reactivation of phosphorylated glutamate dehydrogenase could be inhibited by phosphate, pyrophosphate and fluoride.     

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.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

NAD and NADP-dependent glutamate dehydrogenase in Hydrogenomonas H 16

Summary 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.Low specific activity of NAD-dependent glutamate dehydrogenase was found in cells grown with glutamate as sole nitrogen source or in cells grown with a high concentration of ammonium ions. In the presence of a low concentration of ammonium ions or in a nitrogen free medium, the specific activity of the NAD-dependent enzyme increased. Corresponding to the formation of the NAD-dependent glutamate dehydrogenase the enzyme glutamine synthetase was synthesized. The ratio of NAD-dependent glutamate dehydrogenase to glutamine synthetase activity differed only slightly in cells grown with different nitrogen and carbon sources.The NADP-dependent glutamate dehydrogenase was found in high specific activity in cells grown with an excess of ammonium ions. Under nitrogen starvation the formation of the NADP-dependent glutamate dehydrogenase ceased and the enzyme activity decreased.     

Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group

1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+H(+)), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD(+) and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. The rate of NAD(+) dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).     

Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria

A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD+/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD+ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD+ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I.     

Activation of glutamate by gamma-glutamate kinase: formation of gamma-cis-cycloglutamyl phosphate, an analog of gamma-glutamyl phosphate

gamma-Glutamate kinase, the enzyme that catalyzes the first step in the pathway from glutamate to proline, has been postulated to convert glutamate to a gamma-activated form (possibly gamma-glutamyl phosphate), which is reduced by a NADPH-linked reductase to yield glutamate gamma-semialdehyde (in equilibrium with delta 1-pyrroline-5-carboxylate). In the present work we found that the kinase, in the absence or presence of the reductase (and in the absence of NADPH), catalyzes stoichiometric formation of 5-oxo-L-proline and Pi from L-glutamate and ATP, but catalyzes hydroxamate formation at only about 10% of the rate of ATP-cleavage. A new substrate of the kinase was found; thus, cis-cycloglutamate (cis-1-amino-1,3-dicarboxycyclohexane), a glutamate analog which cannot cyclize to form an analog of 5-oxoproline, interacts effectively with the kinase. The trans form of cycloglutamate does not interact with the kinase; only the cis form can assume a diequatorial conformation equivalent to the extended conformation of glutamate. cis-Cycloglutamyl phosphate formation was shown and evidence was obtained for formation of an enzyme-ADP-cycloglutamyl phosphate complex. Although cis-cycloglutamyl phosphate is not a reducible substrate of the NADPH-dependent reductase, the findings indicate that it interacts with the reductase. These studies, which elucidate several aspects of the mechanism of the utilization of glutamate for formation of delta 1-pyrroline-5-carboxylate, support the hypothesis that the kinase and reductase function as an enzyme complex. A model is suggested in which gamma-glutamyl phosphate formed on the kinase interacts with the reductase to form a gamma-glutamyl-reductase complex, which is reduced by NADPH to yield glutamate gamma-semialdehyde.     

Nitrogen metabolism in Aspergillus parasiticus NRRL 3240 and A. flavus NRRL 3537 in relation to aflatoxin production

The relationship between nitrogen assimilation, metabolism and aflatoxin formation has been investigated in a toxigenic and a non-toxigenic strain of Aspergillus parasiticus. Ammonia from the medium is mainly assimilated via NADP-requiring glutamate dehydrogenase. During growth NAD-requiring glutamate dehydrogenase followed an inverse pattern of activity with respect to NADP glutamate dehydrogenase. Alpha-ketoglutarate, the product of NAD glutamate dehydrogenase, stimulated acetate incorporation into aflatoxins. Glutamine synthetase, ornithine transcarbamylase, both utilizing glutamate as substrate were assayed under different growth conditions. An important regulatory role for glutamine synthetase is suggested. The metabolic route of asparagine utilization was also investigated. Both the known pathways, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase are operative simultaneously.     

Nitrogen metabolism in Aspergillus parasiticus NRRL 3240 and A. flavus NRRL 3537 in relation to aflatoxin production

The relationship between nitrogen assimilation, metabolism and aflatoxin formation has been investigated in a toxigenic and a non-toxigenic strain of Aspergillus parasiticus. Ammonia from the medium is mainly assimilated via NADP-requiring glutamate dehydrogenase. During growth NAD-requiring glutamate dehydrogenase followed an inverse pattern of activity with respect to NADP glutamate dehydrogenase. Alpha-ketoglutarate, the product of NAD glutamate dehydrogenase, stimulated acetate incorporation into aflatoxins. Glutamine synthetase, ornithine transcarbamylase, both utilizing glutamate as substrate were assayed under different growth conditions. An important regulatory role for glutamine synthetase is suggested. The metabolic route of asparagine utilization was also investigated. Both the known pathways, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase are operative simultaneously.     

Action of diclofenac on kidney mitochondria and cells

The mitochondrial membrane potential measured in isolated rat kidney mitochondria and in digitonin-permeabilized MDCK type II cells pre-energized with succinate, glutamate, and/or malate was reduced by micromolar diclofenac dose-dependently. However, ATP biosynthesis from glutamate/malate was significantly more compromised compared to that from succinate. Inhibition of the malate-aspartate shuttle by diclofenac with a resultant decrease in the ability of mitochondria to generate NAD(P)H was demonstrated. Diclofenac however had no effect on the activities of NADH dehydrogenase, glutamate dehydrogenase, and malate dehydrogenase. In conclusion, decreased NAD(P)H production due to an inhibition of the entry of malate and glutamate via the malate-aspartate shuttle explained the more pronounced decreased rate of ATP biosynthesis from glutamate and malate by diclofenac. This drug, therefore affects the bioavailability of two major respiratory complex I substrates which would normally contribute substantially to supplying the reducing equivalents for mitochondrial electron transport for generation of ATP in the renal cell.     

Enzymatic in situ analysis by 1H-NMR of the hydrogen transfer stereospecificity of NAD(P)+-dependent dehydrogenases

We have established a simple procedure for the in situ analysis of stereospecificity of an NAD(P)-dependent dehydrogenase for C-4 hydrogen transfer of NAD(P)H by means of glutamate racemase [EC 5.1.13] and glutamate dehydrogenase [EC 1.4.1.3]. Glutamate racemase inherently catalyzes the exchange of alpha-H of glutamate with 2H during racemization in 2H2O. When the reactions of glutamate racemase and glutamate dehydrogenase, which is pro-S specific for the C4-H transfer of NAD(P)H, are coupled in 2H2O, [4S-2H]-NAD(P)H is exclusively produced. Therefore, if 1H is fully retained at C-4 of NAD(P)+ after incubation of a reaction mixture containing both the enzymes and a dehydrogenase to be tested, the stereospecificity of the dehydrogenase is the same as that of glutamate dehydrogenase. When the C4-H of NAD(P)+ is exchanged with 2H, the enzyme to be examined is different from glutamate dehydrogenase in stereospecificity. Thus, we can readily determine the stereospecificity by 1H-NMR measurement of NAD(P)+ without isolation of the coenzymes and products.     

Enzymatic Regeneration of NAD in Enantioselective Oxidation of Secondary Alcohols: Glutamate Dehydrogenase Versus NADH Dehydrogenase

To improve yield and productivity of ketose in NAD-dependent polyol oxidations, two enzymatic methods for regeneration of the oxidized coenzyme form have been compared and partly optimized for the batch conversion of xylitol into D-xylulose and D-sorbitol into D-fructose. Polyol oxidation was catalyzed by xylitol dehydrogenase from the yeast Galactocandida mastotermitis. Reduction of OM₂ (apparently to H₂O) by partially purified NADH dehydrogenase complex from Corynebacterium callunae could drive alcohol oxidations better than reductive amination of EaL-ketoglutarate by glutamate dehydrogenase. A fed-batch procedure was developed that overcame inhibition of glutamate dehydrogenase by α-ketoglutarate (K_is 25 mM), thus increasing the productivity of ketose almost 2-fold. For D-fructose production from D-sorbitol (0.1-0.3M) yields of < 90% and productivities up to 1.30g/(L.h) have been obtained. High conversion of up to 50g/L xylitol into D-xylulose for which xylitol dehydrogenase exhibits an about 80-fold higher specificity constant than for D-fructose required complexation of the ketose product with borate. In comparison with reductive amination by glutamate dehydrogenase, advantages of using NADH-dehydrogenase catalyzed regeneration of NAD for ketose production are (i) avoidance of byproduct formation, (ii) cheaper substrate (02 versus α-ketoglutarate), and (iii) easier process control (batch versus fed-batch).     

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.     

Regulation of NAD-dependent glutamate dehydrogenase by protein kinases in Saccharomyces cerevisiae

The active NAD-dependent glutamate dehydrogenase of wild type yeast cells fractionated by DEAE-Sephacel chromatography was inactivated in vitro by the addition of either the cAMP-dependent or cAMP-independent protein kinases obtained from wild type cells. cAMP-dependent inhibition of glutamate dehydrogenase activity was not observed in the crude extract of bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase. The cAMP-dependent protein kinase of CYR3 mutant cells, which has a high K alpha value for cAMP in the phosphorylation reaction, required a high cAMP concentration for the inactivation of NAD-dependent glutamate dehydrogenase. An increased inactivation of partially purified active NAD-dependent glutamate dehydrogenase (Mr = 450,000) was observed to correlate with increased phosphorylation of a protein subunit (Mr = 100,000) of glutamate dehydrogenase. The phosphorylated protein was labeled by an NADH analog, 5'-p-fluorosulfonyl[14C]benzoyladenosine. Activation and dephosphorylation of inactive NAD-dependent glutamate dehydrogenase fractions were observed in vitro by treatment with bovine alkaline phosphatase or crude yeast cell extracts. These results suggested that the conversion of the active form of NAD-dependent glutamate dehydrogenase to an inactive form is regulated by phosphorylation through cAMP-dependent and cAMP-independent protein kinases.     

Interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled by fluorescein-5'-isothiocyanate: evidence that the dye participates in the interaction

An interaction of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled with FITC was studied by following the changes in fluorescence intensity of the bound dye. The association between the two enzymes was found to be a rather slow process characterized by a second order rate constant of 1.1 +/- 0.2.10(3) M-1 s-1, the KD of the complex between apoenzymes being 3.2.10(-7) M. The stability of the complex increased upon increase of temperature and ionic strength of the medium, suggesting a hydrophobic character of association. The ligands which bind at the active centers of the two enzymes (NAD+, ATP, 3-phosphoglycerate) weakened the bienzyme association. Unlabeled 3-phosphoglycerate kinase was unable to displace the FITC-labeled enzyme from the complex. Taken together, the results indicate that interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled by FITC is assisted by the dye, which may bind at nucleotide-binding sites of GPDH. No interaction was observed between the FITC-labeled 3-phosphoglycerate kinase and lactate dehydrogenase, which suggests that protein-protein interaction at specific "recognition" sites may be a prerequisite for the complex formation.     

Regulation of ox liver glutamate dehydrogenase activity by coenzymes

The effects of coenzymes NAD(P) and NAD(P)H on the kinetics of the ox liver glutamate dehydrogenase reaction have been studied. The oxidized coenzymes were shown to activate alpha-ketoglutarate amination at inhibiting concentrations of NADH and NADPH. The reduced coenzymes, NADH and NADPH, inhibit glutamate deamination with both NAD and NADP as coenzymes. The data obtained are discussed in terms of literature data on the mechanisms of the coenzyme effects on the glutamate dehydrogenase activity and are inconsistent with the theory of direct ligand--ligand interactions. It was shown that the peculiarities of the glutamate dehydrogenase kinetics can easily be interpreted in the light of the two state models.     

Properties of partially purified saccharopine dehydrogenase from human placenta.

Saccharopine dehydrogenase was previously purified 380-fold from human placenta. The enzyme was shown to catalyze the formation of α-aminoadipic-δ-semialdehyde and glutamate from saccharopine, to have a molecular weight of 480,000 on gel filtration, and not to be separable from l-lysine-α-ketoglutarate reductase. Additional properties of the saccharopine dehydrogenase are now described. The pH optimum for the conversion of saccharopine to glutamate and α-aminoadipic-δ-semialdehyde is 8.5 in Tris-HCl buffer and 8.9 in 2-amino-2-methyl-1,3-propanediol buffer. The specificity of the enzyme for Saccharopine and NAD and the inhibition by glutamate and product analogs were tested. It was found the NADP was the only cofactor that could replace NAD in the enzyme reaction and that several NAD analogs were reaction inhibitors. Glutamate was found to be only moderately effective as an inhibitor. Initial velocity studies revealed that the enzyme has an ordered reaction mechanism. The true K_m values for saccharopine and NAD are 1.15 mm and 0.0645 mm, respectively.     

Studies on associations of glycolytic and glutaminolytic enzymes in MCF-7 cells: Role of P36

Isoelectric focusing of MCF-7 cell extracts revealed an association of the glycolytic enzymes glyceraldehyde 3-phosphate-dehydrogenase, phosphoglycerate kinase, enolase, and pyruvate kinase. This complex between the glycolytic enzymes is sensitive to RNase. p36 could not be detected within this association of glycolytic enzymes; however an association of p36 with a specific form of malate dehydrogenase was found. In MCF-7 cells three forms of malate dehydrogenase can be detected by isoelectric focusing: the mitochondrial form with an isoelectric point between 8.9 and 9.5, the cytosolic form with pl 5.0, and a p36-associated form with pl 7.8. The mitochondrial form comprises the mature mitochondrial isoenzyme (pl 9.5) and its precursor form (pl 8.9). Refocusing of the pl 7.8 form of malate dehydrogenase also gave rise to the mitochondrial isoenzyme. Thus, the pl 7.8 form of malate dehydrogenase is actually the mitochondrial isoenzyme retained in the cytosol by the association with p36. Addition of fructose 1,6-bisphosphate to the initial focusing column induced a quantitative shift of the pl 7.8 form of malate dehydrogenase to the mitochondrial forms (pl 8.9 and 9.5). In MCF-7 cells p36 is not phosphorylated in tyrosine. Kinetic measurements revealed that the pl 7.8 form of malate dehydrogenase has the lowest affinity for NADH. Compared to both mitochondrial forms the cytosolic isoenzyme has a high capacity when measured in the NAD → NADH direction (malate → oxaloacetate direction). The association of p36 with the mitochondrial isoenzyme may favor the flow of hydrogen from the cytosol into the mitochondria. Inhibition of cell proliferation by AMP which leads to an inhibition of glycolysis has no effect on complex formation by glycolytic and glutaminolytic enzymes in MCF-7 cells. AMP treatment leads to an activation of malate dehydrogenase, which correlates with the increase of pyruvate and the decrease of lactate levels, but has no effect on the distribution of the various malate dehydrogenase forms. © 1996 Wiley-Liss, Inc.