Глютаматдегидрогеназа 1 (GLUD1): структура, функция и эволюция.

Enzyme

GLUD1 (glutamate dehydrogenase 1) is a mitochondrial matrix enzyme, one of the family of glutamate dehydrogenases that are ubiquitous in life, with a key role in nitrogen and glutamate (Glu) metabolism and energy homeostasis. This dehydrogenase is expressed at high levels in liver, brain, pancreas and kidney, but not in muscle. In the pancreatic cells, GLUD1 is thought to be involved in insulin secretion mechanisms. In nervous tissue, where glutamate is present in concentrations higher than in the other tissues, GLUD1 appears to function in both the synthesis and the catabolism of glutamate and perhaps in ammonia detoxification.

Human GLUD1 contains 13 exons and is located on the 10th chromosome. There is evidence that GLUD1 has been retro posed to the X chromosome, where it gave rise to the intronless GLUD2 through random mutations and natural selection. GLUD2 have adapted to the particular needs of the nervous system where it is specifically expressed.

GLUD1 is a hexamer. The monomer unit has:
N terminal Glu BD(Binding domain) that is composed mostly of β strands. NAD BD can bind either NAD+ or NADP+. 48 residue antenna like projection that extends from the top of each NAD BD. The antenna consists of an ascending helix and a descending random coil strand that contains a small α helix toward the C terminal end of the strand. NAD BD sits on the top of Glu BD. NAD BD and Glu BD form the catalytic cleft. During substrate binding, the NAD BD moves significantly. This movement has two components, rotating along the long axis of a helix at the back of the NAD BD, called "the pivot helix", and twisting about the antenna in a clockwise fashion. A comparison of the open and closed conformations of GLUD1 reveals changes in the small helix of the descending strand of the antenna, which seems to recoil as the catalytic cleft opens. Closure of one subunit is associated with distortion of the small helix of the descending strand that is pushed into the antenna of the adjacent subunit. R496 is located on this small helix (see Mutations). The core structure of the hexamer is a stacked dimer of trimers. Glu BDs of the monomers are mainly responsible in the buildup of the core. The relative position of the monomers is such that the rotation about the pivot helix in each monomer is not restricted. The antennae from three subunits within the trimers wrap around each other and undergo conformational changes as the catalytic cleft opens and closes. The antenna serves as an intersubunit communication conduit during negative cooperativity and allosteric regulation. Alignment of GLUD1 from various sources, shows that the antenna probably evolved in the protista prior to the formation of purine regulatory sites. This suggests that there is some selective advantage of the antenna itself and that animals evolved new functions for GLUD1 through the addition of allosteric regulation. GLUD1 can form long fibers by end to end association of the hexamers. The polymerization is unrelated to the catalytic activity, but probably has an important role such as formation of multienzyme complexes. GLUD1 has two co enzyme binding sites: one in the NAD BD that is able to bind ether NAD+ or NADP+ and is directly involved in the catalytic process, and a second one, that has regulatory function, lying directly under the pivot helix, that can bind ADP, NAD+, or NADH, but does not bind NADPH well.

GLUD1 catalyses the oxidative deamination of Glu to 2 oxoglutarate and free NH4+ using either NAD+ or NADP+ as a co factor. The reaction occurs with the transfer of a hydride ion from Glu's Cα to NAD(P)+, thereby forming 2 iminoglutarate, which is hydrolyzed to 2 oxoglutarate and NH4+. The reaction's equilibrium under standard circumstances greatly favors Glu formation over NH4+ (Go' ~ 30 kJ. mol 1) formation. For this reason, it was thought that the enzyme played an important role in ammonia detoxification, because since high [NH4+] are toxic, this equilibrium position would be physiologically important; it would help to maintain low [NH4+]. However, in individuals with a certain form of hyperammonemia resulting from a form of hyperinsulinism, the enzyme's activity is increased due to decreased GTP sensitivy, a negative regulator. These individual's blood ammonia levels are raised significantly, which would not be expected if the enzyme did indeed operate at equilibrium.

ADP binds behind the NAD BD, just beneath the pivot helix in the special ADP allosteric site. The adenosine moiety binds down into a hydrophobic pocket, with the ribose phosphate groups pointing outside towards the GTP allosteric site. NADH can also bind to the ADP site, when at high concentrations, usually resulting in the enzyme inhibition.

GTP binding is antagonized by Pi and ADP but is synergistic with NADH bound in the noncatalytic allosteric site. The majority of the contacts between GTP and the enzyme are via the triphosphate moiety. The GTP binding site is considered to be the "sensor" that turns the enzyme off when the cell is at a high energy state. GTP binds at the junction between the NAD BD and the antenna. Whereas most of the GLUD1 GTP interactions are via β and γ phosphate interactions, there are specific interactions with E346 and K343 that favour guanosine over adenosine. In the open conformation, the GTP binding site is distorted such that it can no longer bind GTP. thus allowing GLUD1 to reconcile the non catalytic abortive complexes.

GTP inhibits enzyme turnover over a wide range of conditions by increasing the affinity of GLUD1 for the reaction product, making product release rate limiting under all conditions in the presence of GTP. GTP acts by keeping the catalytic cleft in a closed conformation thus stabilizing the abortive complexes. GTP effects on GLUD1 are not localized solely to the subunit to which it is binding and that the antenna plays an important role in communicating this inhibition to other subunits.

Leu activates GLUD1 independently of the ADP, possessing a special allosteric site in the subunits interface area 8AR7.

FHI is characterized by hypoglycemia that ranges from severe neonatal onset, difficult to manage disease to childhood onset disease with mild symptoms and difficult to diagnose hypoglycemia. Neonatal onset disease manifests within hours to two days after birth. Childhood onset disease manifests during the first months or years of life. In the newborn period, presenting symptoms may be nonspecific, including seizures, hypotonia, poor feeding, and apnea. In severe cases, serum glucose concentrations are typically extremely low and thus easily recognized, whereas in milder cases, variable and mild hypoglycemia may make the diagnosis more difficult. Even within the same family, disease manifestations can range from mild to severe.

Approximately 45% of affected individuals have mutations in either ABCC8, which encodes the protein SUR1, or KCNJ11, which encodes the protein Kir6.2. In the Ashkenazi Jewish population, two ABCC8 founder mutations are responsible for approximately 97% of FHI. Other ABCC8 founder mutations are present in the Finnish population (p. Val187Asp and p. Asp1506Lys). Mutations in GLUD1 and HNF4A each account for approximately 5% of individuals with FHI. Activating mutations in GCK or inactivating mutations in HADH occur in fewer than 1% of individuals with FHI. Mutations in UCP2 have been reported in only two families to date. Approximately 40% of individuals with FHI do not have an identifiable mutation in any of the genes known to be associated with FHI.

At initial diagnosis, hypoglycemia is corrected with intravenous glucose to normalize plasma glucose concentration and prevent brain damage. Long term medical management includes the use of diazoxide, somatostatin analogs, nifedipine, glucagon, recombinant IGF I, glucocorticoids, human growth hormone, dietary intervention, or combinations of these therapies. In individuals in whom aggressive medical management fails to maintain plasma glucose concentration within safe limits, or in whom such therapy cannot be safely maintained over time, pancreatic resection is considered.