Glutamate dehydrogenase (GDH) is a hexameric enzyme that catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia while reducing NAD(P)+ to NAD(P)H. It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate), lipid biosynthesis (via oxidative generation of citrate), and lactate production. While most mammals possess a single GDH1 protein (hGDH1 in the human) that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. The novel hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1) is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH) in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth. In addition, deregulation of hGDH1/2 is implicated in the pathogenesis of several human disorders.

Human glutamate dehydrogenase (GDH) exists in two isoforms encoded by the GLUD1 and GLUD2 genes, respectively. Although the two enzymes share in their mature form all but 15 of their 505 amino acids, they differ markedly in their allosteric regulation. To identify the structural basis for these allosteric characteristics, we performed site-directed mutagenesis on the human GLUD1 gene at sites that differ from the GLUD2 gene using a cloned GLUD1 cDNA. Results showed that substitution of Ala for Gly-456, but not substitution of His for Arg-470 or Ser for Asn-498, renders the enzyme markedly resistant to GTP inhibition (IC 50 ‫؍‬ 2.80 M) as compared with the wild type GLUD1-derived GDH (IC 50 ‫؍‬ 0.19 M). The G456A mutation abolished the cooperative behavior of the enzyme, as revealed by the GTP inhibitory curves. The catalytic and kinetic properties of the G456A mutant and its activation by ADP were comparable with those of the wild type GDH. Gly-456 lies in a very tightly packed region of the GDH molecule, and its replacement by Ala may lead to steric clashes with neighboring amino acids. These, in turn, may affect the conformational state of the protein that is essential for the allosteric regulation of the enzyme by GTP. Glutamate dehydrogenase (GDH)1 (E.C.1.4.1.3) catalyzes the reversible oxidative deamination of glutamate to ␣-ketoglutarate using NAD(H) or NADP(H) as cofactors (1). The mature GDH protein is composed of six identical subunits consisting of 505 amino acids each. The enzyme is thought to play a key role in cellular metabolism and energy homeostasis (2). In the pancreatic ␤ cells, GDH is thought to be involved in insulin secretion mechanisms, whereas in the nervous system the enzyme may play a role in the metabolism of neurotransmitter glutamate and in neurodegenerative processes (3, 4).GDH in humans exists in two different isoforms: a housekeeping and a nerve tissue-specific isoenzyme encoded by the GLUD1 and the GLUD2 gene, respectively (5-8). GLUD1 contains 13 exons and is located on the 10th chromosome, whereas the GLUD2 gene lacks introns and is X-linked. Mammalian GDH is shown to be allosterically regulated by diverse compounds, such as purine nucleotides, steroid hormones, etc (1). GDH regulation is of particular biological importance as exemplified by observations showing that regulatory mutations of the GLUD1 GDH are associated with clinical manifestations in children (9).Although the two GDH isoenzymes are highly homologous (showing a 97% amino acid identity), they differ markedly in their regulatory properties (8, 10). Thus, while the GLUD1-derived GDH is sensitive to GTP inhibition, the GLUD2 GDH is resistant to this compound. In contrast, the GLUD2 GDH is much more sensitive to allosteric activation by ADP or L-leucine than the GLUD1-derived enzyme (10). In addition, there are significant differences between the two isoforms with respect to the K m values for the substrates of the enzyme.Because the GLUD1-and GLUD2-derived polypeptides share in their mature form all but 15 of their 5...

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate using NAD or NADP as cofactors. In mammalian brain, GDH is located predominantly in astrocytes, where it is probably involved in the metabolism of transmitter glutamate. The exact mechanisms that regulate glutamate fluxes through this pathway, however, have not been fully understood. In the human, GDH exists in heat-resistant and heat-labile isoforms, encoded by the GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) genes, respectively. These forms differ in their catalytic and allosteric properties. Kinetic studies showed that the K(m) value for glutamate for the nerve tissue GDH is within the range of glutamate levels in astrocytes (2.43 mM), whereas for the housekeeping enzyme, this value is significantly higher (7.64 mM; P < 0.01). The allosteric activators ADP (0.1-1.0 mM) and L-leucine (1.0-10.0 mM) induce a concentration-dependent enzyme stimulation that is proportionally greater for the nerve tissue-specific GDH (up to 1,600%) than for the housekeeping enzyme (up to 150%). When used together at lower concentrations, ADP (10-50 mM) and L-leucine (75-200 microM) act synergistically in stimulating GDH activity. GTP exerts a powerful inhibitory effect (IC(50) = 0.20 mM) on the housekeeping GDH; in contrast, the nerve tissue isoenzyme is resistant to GTP inhibition. Thus, although the housekeeping GDH is regulated primarily by GTP, the nerve tissue GDH activity depends largely on available ADP or L-leucine levels. Conditions associated with enhanced hydrolysis of ATP to ADP (e.g., intense glutamatergic transmission) are likely to activate nerve tissue-specific GDH leading to an increased glutamate flux through this pathway.

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Human glutamate dehydrogenase (GDH) exists in GLUD1 (housekeeping) and in GLUD2-specified (brainspecific) isoforms, which differ markedly in their basal activity and allosteric regulation. To determine the structural basis of these functional differences, we mutagenized the GLUD1 GDH at four residues that differ from those of the GLUD2 isoenzyme.