Glutaminase Reaction Research Articles

Glutaminases regulate glutathione and oxidative stress in cancer.

Targeted therapies against cancer have improved both survival and quality of life of patients. However, metabolic rewiring evokes cellular mechanisms that reduce therapeutic mightiness. Resistant cells generate more glutathione, elicit nuclear factor erythroid 2-related factor 2 (NRF2) activation, and overexpress many anti-oxidative genes such as superoxide dismutase, catalase, glutathione peroxidase, and thioredoxin reductase, providing stronger antioxidant capacity to survive in a more oxidative environment due to the sharp rise in oxidative metabolism and reactive oxygen species generation. These changes dramatically alter tumour microenvironment and cellular metabolism itself. A rational design of therapeutic combination strategies is needed to flatten cellular homeostasis and accomplish a drop in cancer development. Context-dependent glutaminase isoenzymes show oncogenic and tumour suppressor properties, being mainly associated to MYC and p53, respectively. Glutaminases catalyze glutaminolysis in mitochondria, regulating oxidative phosphorylation, redox status and cell metabolism for tumour growth. In addition, the substrate and product of glutaminase reaction, glutamine and glutamate, respectively, can work as signalling molecules moderating redox and bioenergetic pathways in cancer. Novel synergistic approaches combining glutaminase inhibition and redox-dependent modulation are described in this review. Pharmacological or genetic glutaminase regulation along with oxidative chemotherapy can help to improve the design of combination strategies that escalate the rate of therapeutic success in cancer patients.

“Pinching” the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage

Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either l-glutamine or exogenous NH3 as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH3 passes through a gate located at the end of a ~25-Å tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH3 transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with “pinching” the tunnel, thereby revealing processes that synchronize the transfer of NH3 from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a “bottleneck” effect. The inability of V60F to use exogenous NH3 was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-l-norleucine. Use of NH2OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH3 gate of CTPS to mediate inter-domain NH3 transport.

Glutaminases.

Mammalian glutaminases catalyze the stoichiometric conversion of L-glutamine to L-glutamate and ammonium ions. In brain, glutaminase is considered the prevailing pathway for synthesis of the neurotransmitter pool of glutamate. Besides neurotransmission, the products of glutaminase reaction also fulfill crucial roles in energy and metabolic homeostasis in mammalian brain. In the last years, new functional roles for brain glutaminases are being uncovered by using functional genomic and proteomic approaches. Glutaminases may act as multifunctional proteins able to perform different tasks: the discovery of multiple transcript variants in neurons and glial cells, novel extramitochondrial localizations, and isoform-specific proteininteracting partners strongly support possible moonlighting functions for these proteins. In this chapter, we present a critical account of essential works on brain glutaminase 80 years after its discovery. We will highlight the impact of recent findings and thoughts in the context of the glutamate/glutamine brain homeostasis.

Identification of the Glutaminase Genes ofAspergillus sojaeInvolved in Glutamate Production during Soy Sauce Fermentation

Glutaminase, an enzyme that catalyzes the conversion of L-glutamine to L-glutamate, enhances the umami taste in soy sauce. The Aspergillus sojae genome contains 10 glutaminase genes. In this study, we estimated that approximately 60% of the glutamate in soy sauce is produced through the glutaminase reaction. To determine which glutaminase is involved in soy sauce glutamate production, we prepared soy sauces using single and multiple glutaminase gene disruptants of A. sojae. The glutamate concentration in soy sauce prepared using the ΔgahA-ΔgahB-ΔggtA-Δgls disruptant was approximately 60% lower than that in the control strain, whereas it was decreased by approximately 20-30% in the ΔgahA-ΔgahB disruptant. However, the glutamate concentration was unchanged in the soy sauces prepared using the ΔgahA-ΔggtA-Δgls and ΔgahB-ΔggtA-Δgls disruptants. These results indicate that four glutaminases are involved in glutamate production in soy sauce, and that the peptidoglutaminase activities of GahA and GahB increase the glutamate concentration in soy sauce.

The Role of Glutamine Synthetase and Glutamate Dehydrogenase in Cerebral Ammonia Homeostasis

In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [(13)N]ammonia have shown that about 25 % of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of α-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [(13)N]ammonia in rat brain is incorporated into glutamate and the α-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/α-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/α-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: L-Aspartate + GTP + H(2)O → Fumarate + GDP + P(i) + NH(3)) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research.

Unidirectional Mechanistic Valved Mechanisms for Ammonia Transport in GatCAB.

Glutamine amidotransferase CAB (GatCAB), a crucial enzyme involved in translational fidelity, catalyzes three reactions: (i) the glutaminase reaction to yield ammonia (NH3 or NH4(+)) from glutamine, (ii) the phosphorylation of Glu-tRNA(Gln), and (iii) the transamidase reaction to convert the phosphorylated Glu-tRNA(Gln) to Gln-tRNA(Gln). In the crystal structure of GatCAB, the two catalytic centers are far apart, and the presence of a hydrophilic channel to transport the molecules produced by the reaction (i) was proposed. We investigated the transport mechanisms of GatCAB by molecular dynamics (MD) simulations and free energy (PMF) calculations. In the MD simulations (in total ∼1.1 μs), the entrance of the previously proposed channel is closed, as observed in the crystal structure. Instead, a novel hydrophobic channel has been identified in this study: Since the newly identified entrance opened and closed repeatedly in the MD simulations, it may act as a gate. The calculated free energy difference revealed the significant preference of the newly identified gate/channel for NH3 transport (∼10(4)-fold). In contrast, with respect to NH4(+), the free energy barriers are significantly increased for both channels due to tight hydrogen-bonding with hydrophilic residues, which hinders efficient transport. The opening of the newly identified gate is modulated by Phe206, which acts as a "valve". For the backward flow of NH3, our PMF calculation revealed that the opening of the gate is hindered by Ala207, which acts as a mechanistic "stopper" against the motion of the "valve" (Phe206). This is the first report to elucidate the detailed mechanisms of unidirectional mechanistic valved transport inside proteins.

Brain glutaminases.

Glutaminase is considered as the main glutamate producer enzyme in brain. Consequently, the enzyme is essential for both glutamatergic and gabaergic transmissions. Glutamine-derived glutamate and ammonia, the products of glutaminase reaction, fulfill crucial roles in energy metabolism and in the biosynthesis of basic metabolites, such as GABA, proteins and glutathione. However, glutamate and ammonia are also hazardous compounds and danger lurks in their generation beyond normal physiological thresholds; hence, glutaminase activity must be carefully regulated in the mammalian brain. The differential distribution and regulation of glutaminase are key factors to modulate the metabolism of glutamate and glutamine in brain. The discovery of novel isoenzymes, protein interacting partners and subcellular localizations indicate new functions for brain glutaminase. In this short review, we summarize recent findings that point consistently towards glutaminase as a multifaceted protein able to perform different tasks. Finally, we will highlight the involvement of glutaminase in pathological states and its consideration as a potential therapeutic target.

Functional and Structural Characterization of Four Glutaminases from Escherichia coli and Bacillus subtilis

Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.

Structure–function studies of glutamate synthases: A class of self‐regulated iron‐sulfur flavoenzymes essential for nitrogen assimilation

Glutamate synthases play with glutamine synthetase an essential role in nitrogen assimilation processes in microorganisms, plants, and lower animals by catalyzing the net synthesis of one molecule of L-glutamate from L-glutamine and 2-oxoglutarate. They exhibit a modular architecture with a common subunit or region, which is responsible for the L-glutamine-dependent glutamate synthesis from 2-oxoglutarate. Here, a PurF- (Type II- or Ntn-) type amidotransferase domain is coupled to the synthase domain, a (beta/alpha)8 barrel containing FMN and one [3Fe-4S]0,+1 cluster, through a approximately 30 angstroms-long intramolecular tunnel for the transfer of ammonia between the sites. In bacterial and eukaryotic GltS, reducing equivalents are provided by reduced pyridine nucleotides thanks to the stable association with a second subunit or region, which acts as a FAD-dependent NAD(P)H oxidoreductase and is responsible for the formation of the two low potential [4Fe-4S]+1,+2 clusters of the enzyme. In photosynthetic cells, reduced ferredoxin is the physiological reductant. This review focus on the mechanism of cross-activation of the synthase and glutaminase reactions in response to the bound substrates and the redox state of the enzyme cofactors, as well as on recent information on the structure of the alphabeta protomer of the NADPH-dependent enzyme, which sheds light on the intramolecular electron transfer pathway between the flavin cofactors.

Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate‐activated glutaminase

Phosphate-activated glutaminase is present at high levels in the cerebellar mossy fiber terminals. The role of this enzyme for the production of glutamate from glutamine in the parallel-fiber terminals is unclear. In order to address this, we used light miroscopic immunoperoxidase and electron microscopic immunogold methods to study the localization of glutamate in rat cerbellar slices incubated with physiological K+ (3 mmol/L) and depolarizing K+ (40 mmol/L) concentrations, and during depolarizing conditions with the addition of glutamine and the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine. During K+-induced depolarization glutamate labeling was redistributed from parallel-fiber terminals to glial cells. The nerve terminal content of glutamate was sustained when the slices were supplied with glutamine, which also reduced the accumulation of glutamate in glia. In spite of glutamine supplementation, the depolarized slices treated with 6-diazo-5-oxo-l-norleucine showed depletion of glutamate from parallel-fiber terminals and accumulation in glial cells. We conclude that cerebellar parallel-fiber terminals contain a glutaminase activity enabling them to synthesize glutamate from glutamine. Our results confirm that this is also true for the mossy fiber terminals. In addition, we show that, like for glutamate, the levels of aspartate in parallel-fiber terminals and GABA in Golgi fiber terminals can be maintained during depolarization if glutamine is present. This process is dependent on the activity of a glutaminase, as it can be inhibited by 6-diazo-5-oxo-l-norleucine, suggesting that the glutaminase reaction is important for glutamine to act as a precursor also for aspartate and GABA. The low levels of the kidney type of glutaminase that previously has been shown to be present in the parallel and Golgi fiber terminals could be sufficient to produce the transmitter amino acids. Alternatively, the amino acids could be produced from the liver type of glutaminase, which is not yet localized on the cellular level, or from an unknown glutminase.

Improving the umami taste of soy sauce by the addition of bacterial γ-glutamyltranspeptidase as a glutaminase to the fermentation mixture

γ-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of γ-glutamyl compounds and transfer of their γ-glutamyl moieties to amino acids and peptides. GGT is able to catalyze the hydrolysis of glutamine (Gln) to glutamic acid (Glu), the so called glutaminase reaction. A salt-tolerant wild-type GGT from Bacillus subtilis and D445A mutant GGT, whose transpeptidation activity was abolished, were added to a soy sauce fermentation mixture to increase the amount of Glu in the product. The concentration of Glu in the soy sauce was 36 mM higher with the addition of wild-type GGT than without. Nine out of ten members of a panel recognized a difference and preferred the taste of the soy sauce fermented with the wild-type GGT. These results indicate that wild-type GGT is competent for use in the fermentation of soy sauce as a glutaminase.

Folate synthesis in plants: purification, kinetic properties, and inhibition of aminodeoxychorismate synthase

pABA (p-aminobenzoate) is a precursor of folates and, besides esterification to glucose, has no other known metabolic fate in plants. It is synthesized in two steps from chorismate and glutamine, the first step being their conversion into glutamate and ADC (4-aminodeoxychorismate). In Escherichia coli, two proteins forming a heterodimeric complex are required for this reaction, but, in plants and lower eukaryotes, a single protein is involved. The Arabidopsis enzyme was expressed in E. coli and was purified to homogeneity. The monomeric enzyme (95 kDa) catalyses two reactions: release of NH3 from glutamine (glutaminase activity) and substitution of NH3 for the hydroxy group at position 4 of chorismate (ADC synthase activity). The kinetic parameters of the plant enzyme are broadly similar to those of the bacterial complex, with K(m) values for glutamine and chorismate of 600 and 1.5 microM respectively. As with the bacterial enzyme, externally added NH3 was a very poor substrate for the plant enzyme, suggesting that NH3 released from glutamine is preferentially channelled to chorismate. The glutaminase activity could operate alone, but the presence of chorismate increased the efficiency of the reaction 10-fold, showing the interdependency of the two domains. The plant enzyme was inhibited by dihydrofolate and its analogue methotrexate, a feature never reported for the prokaryotic system. These molecules were inhibitors of the glutaminase reaction, competitive with respect to glutamine (K(i) values of 10 and 1 microM for dihydrofolate and methotrexate respectively). These findings support the view that the monomeric ADC synthase is a potential target for antifolate drugs.

Gln-tRNAGln Formation from Glu-tRNAGln Requires Cooperation of an Asparaginase and a Glu-tRNAGln Kinase

Gln-tRNA(Gln) is synthesized from Glu-tRNA(Gln) in most microorganisms by a tRNA-dependent amidotransferase in a reaction requiring ATP and an amide donor such as glutamine. GatDE is a heterodimeric amidotransferase that is ubiquitous in Archaea. GatD resembles bacterial asparaginases and is expected to function in amide donor hydrolysis. We show here that Methanothermobacter thermautotrophicus GatD acts as a glutaminase but only in the presence of both Glu-tRNA(Gln) and the other subunit, GatE. The fact that only Glu-tRNA(Gln) but not tRNA(Gln) could activate the glutaminase activity of GatD suggests that glutamine hydrolysis is coupled tightly to transamidation. M. thermautotrophicus GatDE enzymes that were mutated in GatD at each of the four critical asparaginase-active site residues lost the ability to hydrolyze glutamine and were unable to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) when glutamine was the amide donor. However, ammonium chloride rescued the activities of these mutants, suggesting that the integrity of the ATPase and the transferase activities in the mutant GatDE enzymes was maintained. In addition, pyroglutamyl-tRNA(Gln) accumulated during the reaction catalyzed by the glutaminase-deficient mutants or by GatE alone. The pyroglutamyl-tRNA is most likely a cyclized by-product derived from gamma-phosphoryl-Glu-tRNA(Gln), the proposed high energy intermediate in Glu-tRNA(Gln) transamidation. That GatE alone could form the intermediate indicates that GatE is a Glu-tRNA(Gln) kinase. The activation of Glu-tRNA(Gln) via gamma-phosphorylation bears a similarity to the mechanism used by glutamine synthetase, which may point to an ancient link between glutamine synthesized for metabolism and translation.

Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity

GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase. In the GATase domain of the recently published structures of the Escherichia coli and Thermus thermophilus CTP synthases a loop region immediately proceeding amino acid residues forming the oxy-anion hole and named lid L11 is shown for the latter enzyme to be flexible and change position depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy-anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid L11 and we have analyzed their possible role in GTP activation. Characterization of the mutant enzymes R359M, R359P, G360A and G360P indicated that both Arg359 and Gly360 are involved in the allosteric response to GTP binding whereas the E362Q enzyme behaved like wild-type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10- to 50-fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four- to 10-fold increases in K(A) for GTP. The R359M, R359P and G360P also showed no GTP activation of the uncoupled glutaminase reaction whereas the G360A enzyme was about twofold more active than wild-type enzyme. The elevated K(A) for GTP and reduced GTP activation of CTP synthesis of the mutant enzymes are in agreement with a predicted interaction of bound GTP with lid L11 and indicate that the GTP activation of glutamine dependent CTP synthesis may be explained by structural rearrangements around the oxy-anion hole of the GATase domain.

Crystal structure of glutamine amidotransferase from Thermotoga maritima.

Glutamine amidotransferases (GATases) are ubiquitous enzymes that transfer the amide nitrogen of glutamine to a variety of substrates.1 GATases catalyze two separate reactions at two active sites, which are located either on a single polypeptide chain or on different subunits. In the glutaminase reaction, glutamine is hydrolyzed to glutamate and ammonia, which is added to an acceptor substrate in the synthase reaction. Imidazole glycerol phosphate synthase (IGPS) is a GATase that catalyzes the bifurcation step of the histidine and de novo purine biosynthetic pathways. In yeast, IGPS is a single polypeptide,2 whereas in bacteria IGPS activity is effected by a complex of a glutaminase subunit, HisH, and a synthase subunit, HisF.3,4 IGPS is a class I GATase, which is characterized by a Cys–His–Glu catalytic triad. IGPS is a potential target for antibiotic and herbicide development because the histidine pathway does not occur in mammals. Here, we present the crystal structure of the HisH protein from Thermotoga maritima (TmHisH), which complements the known structure of HisF from the same organism (TmHisF)5 [Protein Data Bank (PDB) ID 1THF] and HisF from Pyrobaculum aerophilum6 (PDB ID 1H5Y) and shows high structural similarity with the glutaminase domain of the recently determined structure of IGSP from yeast7 (HIS7) (PDB ID 1JVN).

Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.

γ-glutamyl thioester intermediate in glutaminase reaction catalyzed by Escherichia coli asparagine synthetase B

Glutamine-dependent amidotransferases have been classified into three classes on the basis of conserved residues in the deduced amino acid sequences of their glutamine-hydrolyzing domains or subunits. The family of class I amidotransferases, which includes carbamoyl-phosphate synthetase (CPS) and guanosine-5'-monophosphate synthetase (GMPS), is characterized by a conserved Cys-His dyad that is essential in catalyzing the conversion of glutamine to glutamate. The stoichiometry, saturation behavior, and rates of formation and breakdown of the covalent intermediate isolated in these experiments are consistent with its participation in the normal mechanism of AS-B catalyzed glutamine hydrolysis. The putative F-glutamyl thioester intermediate is the covalent adduct isolated when AS-B is incubated with radiolabeled glutanfine. The conserved glutamate in the glutaminase active site of these enzymes has been shown to have no catalytic role, at least in CPS, suggesting that class I amidotransferases catalyze glutamine hydrolysis in a manner akin to thiol proteases, such as papain.

A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons.

The metabolism of [U-(13)C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co-cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with (13)C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate-glutamine/lactate-alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [(15)N]alanine (1 mM) and [5-(15)N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U-(13)C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5-(15)N]glutamine to 3.3% and [U-(13)C]glutamate generated from [U-(13)C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of alpha-ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [(15)N]alanine. Moreover, in co-cultures, [U-(13)C]alanine labeled glutamate and glutamine equally, whereas [U-(13)C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.

Formation and isolation of a covalent intermediate during the glutaminase reaction of a class II amidotransferase.

Incubation of Escherichia coli asparagine synthetase B (AS-B) with [14C]-L-glutamine gives a covalent adduct that can be isolated. Radiolabeled protein is not observed (i) when the wild-type enzyme is incubated with 6-diazo-5-oxo-L-norleucine (DON) prior to reaction with [14C]glutamine or (ii) when the C1A AS-B mutant is incubated with [14C]-L-glutamine. Both of these alterations eliminate the ability of the enzyme to utilize glutamine but do not affect ammonia-dependent asparagine synthesis. Formation of the covalent adduct therefore depends on the presence of the N-terminal active site cysteine, which has been shown to be essential for glutamine-dependent activity in this and other class II amidotransferases. The amount of covalent adduct exhibits saturation behavior with increasing concentrations of L-glutamine. The maximum observed quantity of this intermediate is consistent with its involvement on the main pathway of glutamine hydrolysis. The chemical properties of the isolable covalent adduct are consistent with those anticipated for the gamma-glutamyl thioester that has been proposed as an intermediate in the AS-B-catalyzed conversion of glutamine to glutamate. The covalent adduct is acid-stable but is labile under alkaline conditions. On the basis of the measured rates of formation and breakdown of this intermediate, it is kinetically competent to participate in the normal catalytic mechanism. These studies represent the first description of a thioester intermediate for any class II amidotransferase and represent an important step in gaining further insight into the kinetic and chemical mechanisms of AS-B.

Nucleotide-dependent Tetramerization of CTP Synthetase from Saccharomyces cerevisiae

The nucleotide-dependent tetramerization of purified native URA7-encoded CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) from the yeast Saccharomyces cerevisiae was characterized. CTP synthetase existed as a dimer in the absence of ATP and UTP. In the presence of saturating concentrations of ATP and UTP, the CTP synthetase protein existed as a tetramer. Increasing concentrations of ATP and UTP caused a dose-dependent conversion of the dimeric species to a tetramer. The kinetics of enzyme tetramerization correlates with the kinetics of enzyme activity. The tetramerization of CTP synthetase was dependent on UTP and Mg2+ ions. ATP facilitated the UTP-dependent tetramerization of CTP synthetase by a mechanism that involved the ATP-dependent phosphorylation of UTP catalyzed by the enzyme. The glutaminase reaction that is catalyzed by the enzyme was not required for enzyme tetramerization. CTP, a potent inhibitor of CTP synthetase activity, did not inhibit the ATP/UTP-dependent tetramerization of the enzyme. Phosphorylation of the purified native CTP synthetase with protein kinase A and protein kinase C facilitated the nucleotide-dependent tetramerization. Dephosphorylation of native CTP synthetase with alkaline phosphatase prevented the nucleotide-dependent tetramerization of the enzyme. This correlated with the inactivation of CTP synthetase activity. Rephosphorylation of the dephosphorylated enzyme with protein kinase A and protein kinase C resulted in a partial restoration of the nucleotide-dependent tetramerization of the enzyme. This tetramerization correlated with the partial restoration of CTP synthetase activity. Taken together, these results indicated that enzyme tetramerization was required for CTP synthetase activity and that enzyme phosphorylation played an important role in the tetramerization and regulation of the enzyme.