GLUD1 is a mitochondrial matrix enzyme

Enzyme

Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

, with a key role in the nitrogen and glutamate (Glu) metabolism

Metabolism

Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...

 and the energy homeostasis

Homeostasis

Homeostasis is the property of a system that regulates its internal environment and tends to maintain a stable, constant condition of properties like temperature or pH...

. GLUD1 is expressed at high levels in liver

Liver

The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion...

, brain

Brain

The brain is the center of the nervous system in all vertebrate and most invertebrate animals—only a few primitive invertebrates such as sponges, jellyfish, sea squirts and starfishes do not have one. It is located in the head, usually close to primary sensory apparatus such as vision, hearing,...

, pancreas

Pancreas

The pancreas is a gland organ in the digestive and endocrine system of vertebrates. It is both an endocrine gland producing several important hormones, including insulin, glucagon, and somatostatin, as well as a digestive organ, secreting pancreatic juice containing digestive enzymes that assist...

 and kidney

Kidney

The kidneys, organs with several functions, serve essential regulatory roles in most animals, including vertebrates and some invertebrates. They are essential in the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid–base balance, and...

, but not in muscle

Muscle

Muscle is a contractile tissue of animals and is derived from the mesodermal layer of embryonic germ cells. Muscle cells contain contractile filaments that move past each other and change the size of the cell. They are classified as skeletal, cardiac, or smooth muscles. Their function is to...

. In the pancreatic cells, GLUD1 is thought to be involved in insulin

Insulin

Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....

 secretion

Secretion

Secretion is the process of elaborating, releasing, and oozing chemicals, or a secreted chemical substance from a cell or gland. In contrast to excretion, the substance may have a certain function, rather than being a waste product...

 mechanisms. In nervous tissue, where Glu is present in concentrations higher than in the other tissues, GLUD1 appears to function in both the synthesis and the catabolism of Glu and perhaps in ammonia

Ammonia

Ammonia is a compound of nitrogen and hydrogen with the formula . It is a colourless gas with a characteristic pungent odour. Ammonia contributes significantly to the nutritional needs of terrestrial organisms by serving as a precursor to food and fertilizers. Ammonia, either directly or...

 detoxification.

Structure

GLUD1 is a hexamer. The monomer unit has:

1. N-terminal Glu-BD(Binding domain) that is composed mostly of β-strands.
2. NAD-BD - can bind either NAD⁺ or NADP⁺.
3. 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 build up 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

Regulatory site

A regulatory site is a site on an allosteric protein to which a modulator molecule binds. A ligand-binding site on a receptor or enzyme distinct from the active site. Allosteric modulators alter enzyme activity by binding to the regulatory site. Also known as an "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

Allosteric regulation

In biochemistry, allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein's allosteric site . Effectors that enhance the protein's activity are referred to as allosteric activators, whereas those that decrease the protein's activity are...

.

GLUD1 can form long fibers by end to end association of the hexamers. The polimerization is unrelated to the catalytic activity, but probably has an important role such as formation of multienzyme comolexes.

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.

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.

Function

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

Hyperammonemia

Hyperammonemia is a metabolic disturbance characterised by an excess of ammonia in the blood. It is a dangerous condition that may lead to encephalopathy and death. It may be primary or secondary....

 resulting from a form of hyperinsulinism

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 one blood glucose level but low or high for another...

, 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.

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 [ADP] has 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 [ATP] - 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 [ATP] - 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 [ATP] - 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.

Phosphate

Phosphate and other bivalent anions stabilize GLUD1. Recent structural studies have shown that phosphate molecules bind to the GTP site.

Gene

Human GLUD1 contains 13 exon

Exon

An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...

s and is located on the 10th chromosome

Chromosome

A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...

.

There is evidence that GLUD1 has been retro-posed to the X chromosome, where it gave rise to the intronless GLUD2 through random mutation

Mutation

In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...

s and natural selection. GLUD2 have adapted to the particular needs of the nervous system where it is specifically expressed.

External links

• GeneReviews/NCBI/NIH/UW entry on Familial Hyperinsulinism