Glutamate dehydrogenase 1
|Glutamate dehydrogenase 1|
PDB rendering based on 1hwx.
|Symbols||; GDH; GDH1; GLUD|
|RNA expression pattern|
GLUD1 (Glutamate dehydrogenase 1) is a mitochondrial matrix enzyme, with a key role in the nitrogen and glutamate (Glu) metabolism and the energy homeostasis. GLUD1 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 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 detoxification.
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 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 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 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 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 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.
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.
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 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 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 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 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(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, is another major allosteric inhibitor of GLUD1.
Phosphate and other bivalent anions stabilize GLUD1. Recent structural studies have shown that phosphate molecules bind to the GTP site.
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.
- Smith TJ, Schmidt T, Fang J, Wu J, Siuzdak G, Stanley CA (May 2002). "The structure of apo human glutamate dehydrogenase details subunit communication and allostery". J. Mol. Biol. 318 (3): 765–77. doi:10.1016/S0022-2836(02)00161-4. PMID 12054821.
- Banerjee S, Schmidt T, Fang J, Stanley CA, Smith TJ (April 2003). "Structural studies on ADP activation of mammalian glutamate dehydrogenase and the evolution of regulation". Biochemistry 42 (12): 3446–56. doi:10.1021/bi0206917. PMID 12653548.
- Smith TJ, Peterson PE, Schmidt T, Fang J, Stanley CA (March 2001). "Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation". J. Mol. Biol. 307 (2): 707–20. doi:10.1006/jmbi.2001.4499. PMID 11254391.
- Peterson PE, Smith TJ (July 1999). "The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery". Structure 7 (7): 769–82. doi:10.1016/S0969-2126(99)80101-4. PMID 10425679.
- George A, Bell JE (December 1980). "Effects of adenosine 5'-diphosphate on bovine glutamate dehydrogenase: diethyl pyrocarbonate modification". Biochemistry 19 (26): 6057–61. doi:10.1021/bi00567a017. PMID 7470450.
- Fang, J; Hsu, BY; MacMullen, CM; Poncz, M; Smith, TJ; Stanley, CA (2002). "Expression, purification and characterization of GLUD1 allosteric regulatory mutations". Biochem J. 363 (Pt 1): 81–7. doi:10.1042/0264-6021:3630081. PMC 1222454. PMID 11903050.
- Shashidharan P, Michaelidis TM, Robakis NK, Kresovali A, Papamatheakis J, Plaitakis A (June 1994). "Novel human glutamate dehydrogenase expressed in neural and testicular tissues and encoded by an X-linked intronless gene". J. Biol. Chem. 269 (24): 16971–6. PMID 8207021.
- GeneReviews/NCBI/NIH/UW entry on Familial Hyperinsulinism
- Glutamate dehydrogenase at the US National Library of Medicine Medical Subject Headings (MeSH)