Heterogeneous Cellular Distribution of Glutamate Dehydrogenase in Brain and in Non-neural Tissues

Mammalian glutamate dehydrogenase (GDH) is an evolutionarily conserved enzyme central to the metabolism of glutamate, the main excitatory transmitter in mammalian CNS. Its activity is allosterically regulated and thought to be controlled by the need of the cell for ATP. While in most mammals, GDH is encoded by a single GLUD1 gene that is widely expressed (housekeeping; hGDH1 in the human), humans and other primates have acquired via retroposition a GLUD2 gene encoding an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. Whereas hGDH1 shows high levels of expression in the liver, hGDH2 is expressed in human testis, brain and kidney. Recent studies have provided significant insight into the functional adaptation of hGDH2. This includes resistance to GTP control, enhanced sensitivity to inhibition by estrogens and other endogenous allosteric effectors, and ability to function in a relatively acidic environment. While inhibition of hGDH1 by GTP, derived from Krebs cycle, represents the main mechanism by which the flux of glutamate through this pathway is regulated, dissociation of hGDH2 from GTP control may provide a biological advantage by permitting enzyme function independently of this energy switch. Also, the relatively low optimal pH for hGDH2 is suited for transmitter glutamate metabolism, as glutamate uptake by astrocytes leads to significant mitochondrial acidification. Although mammalian GDH is a housekeeping enzyme, its levels of expression vary markedly among the various tissues and among the different types of cells that constitute the same organ. In this paper, we will review existing evidence on the cellular and subcellular distribution of GDH in neural and non-neural tissues of experimental animals and humans, and consider the implications of these findings in biology of these tissues. Special attention is given to accumulating evidence that glutamate flux through the GDH pathway is linked to cell signaling mechanisms that may be tissue-specific.     

Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State

Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC₅₀ = 0.1–0.3 μm) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC₅₀ = 0.08 ± 0.01 μm) and with ∼18-fold lower affinity hGDH1 (IC₅₀ = 1.67 ± 0.06 μm; p < 0.001). Similarly, 17β-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC₅₀ = 1.53 ± 0.24 μm) than for hGDH1 (IC₅₀ = 26.94 ± 1.07 μm; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.     

The complex regulation of human glud1 and glud2 glutamate dehydrogenases and its implications in nerve tissue biology

Mammalian glutamate dehydrogenase (GDH) is a housekeeping mitochondrial enzyme (hGDH1 in the human) that catalyses the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, thus interconnecting amino acid and carbohydrate metabolism. It displays an energy sensing mechanism, which permits enzyme activation under low cellular energy states. As GDH is at the crossroads of important metabolic pathways, a tight control of its activity is essential. Indeed, to fulfill its role in metabolism and cellular energetics, mammalian GDH has evolved into a highly regulated enzyme subject to allosteric modulation by diverse compounds. The recent emergence (<23million years ago) in apes and humans of a hGDH2 isoenzyme with distinct regulatory properties, as well as, the detection of gain-of-function variants in hGDH1 and hGDH2 that affect the nervous system, have introduced additional complexities. The properties of the two highly homologous human GDHs were studied using purified recombinant hGDH1 and hGDH2 obtained by expression of the corresponding cDNAs in Sf21 cells. Results showed that, in contrast to hGDH1 that maintains substantial basal activity (35-40% of its maximal capacity), hGDH2 displays low basal activity (3-8% of maximal) that is remarkably responsive to activation by rising levels of ADP and/or l-leucine. This is primarily due to the Arg443Ser evolutionary change, which also made hGDH2 markedly sensitive to estrogens and neuroleptic drugs. In contrast to hGDH1, which is subject to potent GTP inhibition, hGDH2 has dissociated its function from this energy switch, being able to metabolize glutamate even when the Krebs cycle generates GTP levels sufficient to inactivate the housekeeping hGDH1. Our data also show that spermidine, a polyamine thought to reduce oxidative stress and to prolong survival, and EGCG, a green tea polyphenol, inhibit hGDH2 at lower concentrations than hGDH1. The implications of these findings in nerve tissue biology are discussed.     

Inhibitory properties of nerve-specific human glutamate dehydrogenase isozyme by chloroquine

Human glutamate dehydrogenase exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. In the nervous system, GDH is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter during neurotransmission. Chloroquine has been known to be a potent inhibitor of house-keeping GDH1 in permeabilized liver and kidney-cortex of rabbit. However, the effects of chloroquine on nerve-specific GDH2 have not been reported yet. In the present study, we have investigated the effects of chloroquine on hGDH2 at various conditions and showed that chloroquine could inhibit the activity of hGDH2 at dose-dependent manner. Studies of the chloroquine inhibition on enzyme activity revealed that hGDH2 was relatively less sensitive to chloroquine inhibition than house-keeping hGDH1. Incubation of hGDH2 was uncompetitive with respect of NADH and non-competitive with respect of 2-oxoglutarate. The inhibitory effect of chloroquine on hGDH2 was abolished, although in part, by the presence of ADP and L-leucine, whereas GTP did not change the sensitivity to chloroquine inhibition. Our results show a possibility that chloroquine may be used in regulating GDH activity and subsequently glutamate concentration in the central nervous system.     

The human GLUD2 glutamate dehydrogenase and its regulation in health and disease

Whereas glutamate dehydrogenase in most mammals (hGDH1 in the human) is encoded by a single functional GLUD1 gene expressed widely, humans and other primates have acquired through retroposition an X-linked GLUD2 gene that encodes a highly homologous isoenzyme (hGDH2) expressed in testis and brain. Using an antibody specific for hGDH2, we showed that hGDH2 is expressed in testicular Sertoli cells and in cerebral cortical astrocytes. Although hGDH1 and hGDH2 have similar catalytic properties, they differ markedly in their regulatory profile. While hGDH1 is potently inhibited by GTP and may be controlled by the need of the cell for ATP, hGDH2 has dissociated its function from GTP and may metabolize glutamate even when the Krebs cycle generates GTP amounts sufficient to inactivate hGDH1. As astrocytes are known to provide neurons with lactate that largely derives from the Krebs cycle via conversion of glutamate to α-ketoglutarate, the selective expression of hGDH2 may facilitate metabolic recycling processes essential for glutamatergic transmission. As there is evidence for deregulation of glutamate metabolism in degenerative neurologic disorders, we sequenced GLUD1 and GLUD2 genes in neurologic patients and found that a rare T1492G variation in GLUD2 that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 interacted significantly with Parkinson's disease (PD) onset. Thus, in two independent Greek and one North American PD cohorts, Ser445Ala hemizygous males, but not heterozygous females, developed PD 6-13 years earlier than subjects with other genotypes. The Ala445-hGDH2 variant shows enhanced catalytic activity that is resistant to modulation by GTP, but sensitive to inhibition by estrogens. These observations are thought to suggest that enhanced glutamate oxidation by the Ala445-hGDH2 variant accelerates nigral cell degeneration in hemizygous males and that inhibition of the overactive enzyme by estrogens protects heterozygous females. We then evaluated the interaction of estrogens and neuroleptic agents (haloperidol and perphenazine) with the wild-type hGDH1 and hGDH2 and found that both inhibited hGDH2 more potently than hGDH1 and that the evolutionary Arg443Ser substitution was largely responsible for this sensitivity. Hence, the properties acquired by hGDH2 during its evolution have made the enzyme a selective target for neuroactive steroids and drugs, providing new means for therapeutic interventions in disorders linked to deregulation of this enzyme.     

The Discovery of Human of GLUD2 Glutamate Dehydrogenase and Its Implications for Cell Function in Health and Disease

While the evolutionary changes that led to traits unique to humans remain unclear, there is increasing evidence that enrichment of the human genome through DNA duplication processes may have contributed to traits such as bipedal locomotion, higher cognitive abilities and language. Among the genes that arose through duplication in primates during the period of increased brain development was GLUD2, which encodes the hGDH2 isoform of glutamate dehydrogenase expressed in neural and other tissues. Glutamate dehydrogenase GDH is an enzyme central to the metabolism of glutamate, the main excitatory neurotransmitter in mammalian brain involved in a multitude of CNS functions, including cognitive processes. In nerve tissue GDH is expressed in astrocytes that wrap excitatory synapses, where it is thought to play a role in the metabolic fate of glutamate removed from the synaptic cleft during excitatory transmission. Expression of GDH rises sharply during postnatal brain development, coinciding with nerve terminal sprouting and synaptogenesis. Compared to the original hGDH1 (encoded by the GLUD1 gene), which is potently inhibited by GTP generated by the Krebs cycle, hGDH2 can function independently of this energy switch. In addition, hGDH2 can operate efficiently in the relatively acidic environment that prevails in astrocytes following glutamate uptake. This adaptation is thought to provide a biological advantage by enabling enhanced enzyme catalysis under intense excitatory neurotransmission. While the novel protein may help astrocytes to handle increased loads of transmitter glutamate, dissociation of hGDH2 from GTP control may render humans vulnerable to deregulation of this enzyme’s function. Here we will retrace the cloning and characterization of the novel GLUD2 gene and the potential implications of this discovery in the understanding of mechanisms that permitted the brain and other organs that express hGDH2 to fine-tune their functions in order to meet new challenging demands. In addition, the potential role of gain-of-function of hGDH2 variants in human neurodegenerative processes will be considered.     

Glutamate Dehydrogenase Isoforms with N-Terminal (His)6- or FLAG-Tag Retain Their Kinetic Properties and Cellular Localization

Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)₆ small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)₆ tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni²⁺-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)₆ tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5′-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)₆ tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.     

Evolution of <Emphasis Type="Italic">GLUD2</Emphasis> Glutamate Dehydrogenase Allows Expression in Human Cortical Neurons

Human hGDH2 arose via duplication in the apes and driven by positive selection acquired enhanced catalytic ability under conditions inhibitory to its precursor hGDH1 (common to all mammals). To explore the biological advantage provided by the novel enzyme, we studied, by immunohistochemistry (IHC) and immunofluorescence (IF), hGDH1 and hGDH2 expression in the human brain. Studies on human cortical tissue using anti-hGDH1-specific antibody revealed that hGDH1 was expressed in glial cells (astrocytes, oligodendrocytes, and oligodendrocyte precursors) with neurons being devoid of hGDH1 staining. In contrast, an hGDH2-specific antiserum labeled both astrocytes and neurons. Specifically, hGDH2 immunoreactivity was found in the cytoplasm of large neuronal cells within coarse structures resembling mitochondria. These were distributed either in the perikaryon or in the cell periphery. Double immunofluorescence (IF) suggested that the latter represented hGDH2-labeled mitochondria of presynaptic nerve terminals. Hence, hGDH2 evolution bestowed large human neurons with enhanced glutamate metabolizing capacity, thus strengthening cortical excitatory transmission.     

Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes

Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45 degrees C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half-life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 degrees C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.     

The Odyssey of a Young Gene: Structure–Function Studies in Human Glutamate Dehydrogenases Reveal Evolutionary-Acquired Complex Allosteric Regulation Mechanisms

Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35–40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or l-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have performed mutagenesis at sites of difference in their amino acid sequence. Results showed that the low basal activity, heat-lability and estrogen sensitivity of hGDH2 could be, at least partially, ascribed to the Arg443Ser evolutionary change, whereas resistance to GTP inhibition has been attributed to the Gly456Ala change. Other amino acid substitutions studied thus far cannot explain all the remaining functional differences between the two iso-enzymes. Also, the Arg443Ser/Gly456Ala double mutation in hGDH1 approached the properties of wild-type hGDH2, without being identical to it. The insights into the structural mechanism of enzymatic regulation and the implications in cell biology provided by these findings are discussed.     

Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms: implications for biologic function

Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM:, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by approximately 1,600% and that of the GLUD1-derived GDH by approximately 75%. Concentrations of L-leucine similar to those present in human tissues ( approximately 0.1 mM:) had little effect on either isoenzyme. However, the presence of ADP (10-50 microM:) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC(50) = 0.20 microM:), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC(50) > 5,000 microM:). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the K:(m) values obtained for alpha-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.     

Identification of ADP-ribosylation site in human glutamate dehydrogenase isozymes

When the influence of ADP-ribosylation on the activities of the purified human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was measured in the presence of 100 microM NAD+ for 60 min, hGDH isozymes were inhibited by up to 75%. If incubations were performed for longer time periods up to 3 h, the inhibition of hGDH isozymes did not increased further. This phenomenon may be related to the reversibility of ADP-ribosylation in mitochondria. ADP-ribosylated hDGH isozymes were reactivated by Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. The stoichiometry between incorporated ADP-ribose and GDH subunits shows a modification of one subunit per catalytically active homohexamer. Since ADP and GTP had no effects on the extent of modification, it would appear that the ADP-ribosylation is unlikely to occur in allosteric sites. It has been proposed that Cys residue may be involved in the ADP-ribosylation of GDH, although identification of the reactive Cys residue has not been reported. To identify the reactive Cys residue involved in the ADP-ribosylation, we performed cassette mutagenesis at three different positions (Cys59, Cys119, and Cys274) using synthetic genes of hGDH isozymes. Among the Cys residues tested, only Cys119 mutants showed a significant reduction in the ADP-ribosylation. These results suggest a possibility that the Cys119 residue has an important role in the regulation of hGDH isozymes by ADP-ribosylation.     

Critical role of the cysteine 323 residue in the catalytic activity of human glutamate dehydrogenase isozymes

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the Km of the enzyme for NADH and alpha-ketoglutarate increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.     

Study of structure-function relationships in human glutamate dehydrogenases reveals novel molecular mechanisms for the regulation of the nerve tissue-specific (GLUD2) isoenzyme

In mammalian brain, glutamate dehydrogenase (GDH) is located predominantly in astrocytes, where is thought to play a role in transmitter glutamate's metabolism. Human GDH exists in GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) isoforms, which share all but 15 out of their 505 amino acids. The GLUD1 GDH is potently inhibited by GTP, whereas the GLUD2 enzyme is resistant to this compound. On the other hand, the GLUD2 isoform assumes in the absence of GTP a conformational state associated with little catalytic activity, but it remains amenable to full activation by ADP and/or L-leucine. Site-directed mutagenesis of the GLUD1 gene at sites that differ from the corresponding residues of the GLUD2 gene showed that replacement of Gly456 by Ala made the enzyme resistant to GTP (IC(50)=2.8+/-0.15 microM) compared to the wild-type GDH (IC(50)=0.19+/-0.01 microM). In addition, substitution of Ser for Arg443 virtually abolished basal activity and rendered the enzyme dependent on ADP for its function. These properties may permit the neural enzyme to be recruited under conditions of low energy charge (high ADP:ATP ratio), similar to those that prevail in synaptic astrocytes during intense glutamatergic transmission. Hence, substitution of Ser for Arg443 and Ala for Gly456 are the main evolutionary changes that led to the adaptation of the GLUD2 GDH to the unique metabolic needs of the nerve tissue.     

Nerve Tissue-Specific Human Glutamate Dehydrogenase that Is Thermolabile and Highly Regulated by ADP

Abstract: Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH-specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP. However, in the absence of ADP, the nerve tissue-specific GDH showed only 5% of its maximal activity, compared with ～40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05–0.25 mM) induced a concentration-dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550–1,300%) than of the housekeeping enzyme (by 120–150%). Magnesium chloride (1–2 mM) inhibited the nonactivated housekeeping GDH (by 45–64%); this inhibition was reversed almost completely by ADP. In contrast, Mg²⁺ did not affect the nonstimulated nerve tissue-specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05–0.25 mM). Heat-inactivation experiments revealed that the half-life of the housekeeping and nerve tissue-specific GDH was 3.5 and 0.5 h, respectively. Hence, the nerve tissue-specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme. These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands.     

Side‐chain interactions in the regulatory domain of human glutamate dehydrogenase determine basal activity and regulation

Glutamate Dehydrogenase (GDH) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system (CNS). hGDH1 is activated by ADP and L‐leucine and powerfully inhibited by GTP. Besides this housekeeping hGDH1, duplication led to an hGDH2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4–6% of capacity) while remaining remarkably responsive to ADP/L‐leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH1 is shown to diminish basal activity (< 2% of capacity) and abrogate L‐leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain (‘antenna’), we replaced Ser409 by Arg or Asp in hGDH1. The Ser409Arg‐1 change essentially replicated the Arg443Ser‐1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443‐1 or Arg409‐1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser‐1 mutant protein, obtained in recombinant form, regained most of the wild‐type hGDH1 properties. Also, when Ser443 was replaced by Arg443 in hGDH2 (as occurs in hGDH1), the Ser443Arg‐2 mutant acquired most of the hGDH1 properties. Hence, side‐chain interactions between 409 and 443 positions in the ‘antenna’ region of hGDHs are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation.

     

Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is unique in that the enzyme is subject to allosteric activation by leucine. To elucidate the structural basis for leucine-induced allosteric activation of GDH, we determined the crystal structures of the GdhB-Glu and GdhA-GdhB-Leu complexes at 2.1 and 2.6 Å resolution, respectively. The GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits. The GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex. Consistent with the structure, replacement of the amino acid residues of T. thermophilus GDH responsible for leucine binding made T. thermophilus GDH insensitive to leucine. Equivalent amino acid replacement caused a similar loss of sensitivity to leucine in human GDH2, suggesting that human GDH2 also uses the same allosteric site for regulation by leucine.     

Amino acid changes within antenna helix are responsible for different regulatory preferences of human glutamate dehydrogenase isozymes

Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2(390-448))hGDH1 (amino acid segment 390-448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1(390-448))hGDH2 (amino acid segment 390-448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.