GLUD1 (glutamate dehydrogenase 1) is a
mitochondrial
A mitochondrion (; ) is an organelle found in the cells of most Eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is use ...
matrix
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
, one of the family of
glutamate dehydrogenase
Glutamate dehydrogenase (GLDH, GDH) is an enzyme observed in both prokaryotes and eukaryotic mitochondria. The aforementioned reaction also yields ammonia, which in eukaryotes is canonically processed as a substrate in the urea cycle. Typical ...
s that are ubiquitous in
life
Life is a quality that distinguishes matter that has biological processes, such as signaling and self-sustaining processes, from that which does not, and is defined by the capacity for growth, reaction to stimuli, metabolism, energ ...
, with a key role in
nitrogen
Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...
and
glutamate
Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can syn ...
(Glu)
metabolism
Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run cell ...
and energy
homeostasis
In biology, homeostasis (British English, British also homoeostasis) Help:IPA/English, (/hɒmɪə(ʊ)ˈsteɪsɪs/) is the state of steady internal, physics, physical, and chemistry, chemical conditions maintained by organism, living systems. Thi ...
. This
dehydrogenase
A dehydrogenase is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+/NADP+ or a flavin coenzyme such as FAD or FMN. Like all catalysts, they catalyze reverse as well as f ...
is expressed at high levels in
liver
The liver is a major Organ (anatomy), organ only found in vertebrates which performs many essential biological functions such as detoxification of the organism, and the Protein biosynthesis, synthesis of proteins and biochemicals necessary for ...
,
brain
A brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as vision. It is the most complex organ in a v ...
,
pancreas
The pancreas is an organ of the digestive system and endocrine system of vertebrates. In humans, it is located in the abdomen behind the stomach and functions as a gland. The pancreas is a mixed or heterocrine gland, i.e. it has both an end ...
and
kidney
The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; blood ...
, but not in
muscle
Skeletal muscles (commonly referred to as muscles) are organs of the vertebrate muscular system and typically are attached by tendons to bones of a skeleton. The muscle cells of skeletal muscles are much longer than in the other types of muscl ...
. In the pancreatic
cells
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...
, GLUD1 is thought to be involved in
insulin
Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism o ...
secretion 440px
Secretion is the movement of material from one point to another, such as a secreted chemical substance from a cell or gland. In contrast, excretion is the removal of certain substances or waste products from a cell or organism. The classical ...
mechanisms. In nervous tissue, where glutamate is present in concentrations higher than in the other tissues, GLUD1 appears to function in both the
synthesis
Synthesis or synthesize may refer to:
Science Chemistry and biochemistry
*Chemical synthesis, the execution of chemical reactions to form a more complex molecule from chemical precursors
** Organic synthesis, the chemical synthesis of organ ...
and the
catabolism
Catabolism () is the set of metabolic pathways that breaks down molecules into smaller units that are either oxidized to release energy or used in other anabolic reactions. Catabolism breaks down large molecules (such as polysaccharides, lipids, ...
of glutamate and perhaps in
ammonia
Ammonia is an inorganic compound of nitrogen and hydrogen with the formula . A stable binary hydride, and the simplest pnictogen hydride, ammonia is a colourless gas with a distinct pungent smell. Biologically, it is a common nitrogenous was ...
detoxification.
Structure
Gene
Human ''GLUD1'' contains 13
exon
An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding sequen ...
s and is located on the 10th
chromosome
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
.
There is evidence that ''GLUD1'' has been retro-posed to the X chromosome, where it gave rise to the intronless ''GLUD2'' through random
mutation
In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...
s and natural selection. ''GLUD2'' have adapted to the particular needs of the nervous system where it is specifically expressed.
Protein
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 site
In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site.
The site to which the effector binds is termed the ''allosteric site ...
s. 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
In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site.
The site to which the effector binds is termed the ''allosteric site ...
.
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.
Function
GLUD1 catalyses the oxidative deamination of Glu to 2-oxoglutarate and free NH
4+ 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 NH
4+. The reaction's equilibrium under standard circumstances greatly favors Glu formation over NH
4+ (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
4+">H4+are toxic, this equilibrium position would be physiologically important; it would help to maintain low
4+">H4+ However, in individuals with a certain form of
hyperammonemia
Hyperammonemia is a metabolic disturbance characterised by an excess of ammonia in the blood. It is a dangerous condition that may lead to brain injury and death. It may be primary or secondary.
Ammonia is a substance that contains nitrogen. It i ...
resulting from a form of
hyperinsulinism
Hyperinsulinism refers to an above normal level of insulin in the blood of a person or animal. Normal insulin secretion and blood levels are closely related to the level of glucose in the blood, so that a given level of insulin can be normal for on ...
, 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.
Interactions
Binding partners
ADP
ADP binds behind the NAD-BD, just beneath the pivot helix - the second coenzyme binding site. The adenosine moiety binds down into a hydrophobic pocket with the ribose phosphate groups pointing up toward the pivot helix.
ADP can also bind to the second, inhibitory, NADH-site yet causes activation.
GTP
GTP binding is antagonized by P
i 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.
Regulation
When GLUD1 is highly saturated with the active site ligands (substrates), an inhibitory abortive complex forms in the active site: NAD(P)H.Glu in the oxidative deamination reaction at high pH, and NAD(P)
+.2-oxoglutarate in the reductive amination reaction at low pH. GLUD1 assumes its basal state configuration in the absence of allosteric effectors, regardless of whether the allosteric sites are functional. The allosteric regulators of GLUD1 - ADP, GTP, Leu, NAD
+ and NADH - exert their effects by changing the energy required to open and close the catalytic cleft during enzymic turnover, in other words by destabilizing or stabilizing, respectively, the abortive complexes. Activators are not necessary for the catalytic function of GLUD1, as it is active in the absence of these compounds (basal state). It has been suggested that GLUD1 assumes in its basal state a configuration (open catalytic cleft) that permits catalytic activity regardless of whether the allosteric sites are functional. GLUD regulation is of particular biological importance as exemplified by observations showing that regulatory mutations of GLUD1 are associated with clinical manifestations in children.
ADP
ADP being one of the two major activators (NAD
+ being the other one), acts by destabilizing the abortive complexes, and abrogating the negative cooperativity. In the absence of substrates, and with bound ADP, the catalytic cleft is in the open conformation, and the GLUD1 hexamers form long polymers in the crystal cell with more interactions than found in the abortive complex crystals
1NQT. This is consistent with the fact that ADP promotes aggregation in solution. When the catalytic cleft opens, R516 is rotated down on to the phosphates of ADP.
The opening of the catalytic cleft is roughly correlated with distance between R516 and phosphates of ADP. In this way, ADP activates GLUD1 by facilitating the opening of the catalytic cleft which decreases product affinity and facilitates product release.
thus allowing GLUD1 to reconcile the non-catalytic abortive complexes.
Inhibition by high
DPhas been suggested previously to be due to competition between ADP and the adenosine moiety of the coenzyme at the active site1. At least it is known that the effect is relatively unaffected by either H507Y or R516A.
ATP
ATP has complex concentration dependent effects on GLUD1 activity:
* Low
TP- inhibition, mediated through the GTP-binding site, since it is eliminated by H507Y. The affinity of ATP for the GTP site appears to be 1000-fold lower than for GTP, since the β- and γ-phosphate interactions are the major determinant of binding at the GTP site.
* Intermediate
TP- activation, mediated through the ADP effector site, since it is almost completely eliminated by R516A. At this site the nucleotide group is the major determinant of binding.
* High
TP- inhibition, mediated by weak binding at a third site, which is relatively specific for the adenine nucleotides. This effect is relatively unaffected by either H507Y or R516A. As suggested for ADP it could be due to a competition between ATP and the adenosine moiety of the coenzyme at the active site.
GTP
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
Leu activates GLUD1 independently of the ADP site by binding elsewhere, perhaps directly within the catalytic cleft. The enhanced responses of HI/HA patients (see HI/HA syndrom) to Leu stimulation of INS release3, which result from their impaired sensitivity to GTP inhibition, emphasize the physiological importance of inhibitory control of GLUD1.
NAD+
NAD(P)(H) can bind to a second site on each subunit. This site binds NAD(H) ~ 10-fold better than NADP(H) with the reduced forms better than the oxidized forms. Although it has been suggested that binding of the reduced coenzyme at this site inhibits the reaction, while oxidized coenzyme binding causes activation, the effect is still unclear.
NADH
NADH, is another major allosteric inhibitor of GLUD1.
Phosphate
Phosphate and other bivalent anions stabilize GLUD1. Recent structural studies have shown that phosphate molecules bind to the GTP site.
Clinical significance
Familial hyperinsulinism, linked to mutations in GLUD1, 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
Hypoglycemia, also called low blood sugar, is a fall in blood sugar to levels below normal, typically below 70 mg/dL (3.9 mmol/L). Whipple's triad is used to properly identify hypoglycemic episodes. It is defined as blood glucose belo ...
. 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. Individuals with autosomal recessive familial hyperinsulinism, caused by mutations in either
ABCC8
ATP-binding cassette transporter sub-family C member 8 is a protein that in humans is encoded by the ''ABCC8'' gene. ''ABCC8'' orthologs have been identified in all mammals for which complete genome data are available.
The protein encoded by this ...
or
KCNJ11
Kir6.2 is a major subunit of the ATP-sensitive K+ channel, a lipid-gated inward-rectifier potassium ion channel. The gene encoding the channel is called KCNJ11 and mutations in this gene are associated with congenital hyperinsulinism.
Structu ...
(FHI-KATP), tend to be large for gestational age and usually present with severe refractory hypoglycemia in the first 48 hours of life; affected infants usually respond only partially to diet or medical management (i.e., diazoxide therapy) and thus may require pancreatic resection. Individuals with autosomal dominant FHI-
KATP :''KATP is also the ATP-sensitive potassium channel responsible for pancreatic beta-cell insulin release.''
KATP (101.9 FM, "The Bull") is a radio station serving the Amarillo, Texas, metro area with a country music format. It is owned by Townsq ...
tend to be appropriate for gestational age at birth, to present at approximately age one year (range: 2 days - 30 years), and to respond to diet and diazoxide therapy. Exceptions to both of these generalities have been reported. FHI-GCK, caused by mutations in
GCK, may be much milder than FHI-KATP; however, some persons have severe, diazoxide-unresponsive hypoglycemia. FHI-HADH, caused by mutations in HADH, tends to be relatively mild, although severe cases have been reported. Individuals with FHI-HNF4A, caused by mutations in HNF4A, are typically born large for gestational age and have mild features that respond to
diazoxide
Diazoxide, sold under the brand name Proglycem and others, is a medication used to treat low blood sugar due to a number of specific causes. This includes islet cell tumors that cannot be removed and leucine sensitivity. It can also be used in re ...
treatment. FHI-UCP2, caused by mutations in UCP2, is a rare cause of diazoxide-responsive FH1. Hyperammonemia/hyperinsulinism (HA/HI) is associated with mild-to-moderate hyperammonemia and with relatively mild, late-onset hypoglycemia; most but not all affected individuals have mutations in GLUD1.
Clinical characteristics
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.
Diagnosis/testing
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.
Management
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.
References
External links
GeneReviews/NCBI/NIH/UW entry on Familial Hyperinsulinism*
*
{{NLM content
EC 1.4.1