Sandbox Reserved 761

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This Sandbox is Reserved from Sep 25, 2013, through Mar 31, 2014 for use in the course "BCH455/555 Proteins and Molecular Mechanisms" taught by Michael B. Goshe at the North Carolina State University. This reservation includes Sandbox Reserved 299, Sandbox Reserved 300 and Sandbox Reserved 760 through Sandbox Reserved 779.
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Glutamate Dehydrogenase complexed with NADPH, Glutamate, and GTP

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Glutamate DehydrogenaseGlutamate Dehydrogenase

Figure 1. Image of Glutamate Dehydrogenase

Glutamate Dehydrogenase (GDH) is a homohexameric enzyme found in all organisms that catalyses the reversible oxidative deamination of L-glutamate to α-ketoglutarate, and vice versa using NAD+ and/or NADP+ as coenzyme. Located in the mitochondria, GDH plays a key role in urea synthesis, nitrogen and glutamate (Glu) metabolism, and the energy homeostasis. In humans,GDH is expressed at high levels in liver, brain, pancreas and kidney. Acting as an oxidoreductase (Enzyme Class I) , GDH catalyzes the reversible NAD (P)+-linked oxidative deamination of L-glutamate into alpha ketoglutarate and ammonia in two steps. The first step involves a Schiff base intermediate being formed between ammonia and alpha ketoglutarate. This Schiff base intermediate is crucial because it establishes the alpha carbon atom in glutamate’s stereochemistry. The second step involves the Schiff base intermediate being protonated, which is done by the transfer of a hydride ion from NADPH resulting in L-glutamate. GDH is unique because it is able to utilize both NAD+ and NADP+ [1]. NADP+ is utilized in the forward reaction of alpha ketogluterate and free ammonia, which are converted to L-glutamate via a hydride transfer from NADPH to glutamate (15). NAD+ is utilized in the reverse reaction, which involves L-glutamate being converted to alpha ketoglutarate and free ammonia via an oxidative deamination reaction [2]. The extensive production of ammonia by peripheral tissue or glutamate dehydrogenase is not allowed because of the highly toxic effects of circulating ammonia in cells. As a result, the ammonia produced in the reverse reaction of GDH is excreted as NH4+ in the urine, by first going through the urea cycle.

Glutamate Dehydrogenase StructureGlutamate Dehydrogenase Structure

GDH is a homohexamer of 505 residues with a molecular weight of 55.638 KDa [3]. The overall of GDH is composed of eighteen alpha helices and thirteen beta strands, which are both parallel and anti-parallel and flanked by a layer of alpha helices [4]. The monomer unit of GDH is essentially two trimers of six identical subunits containing —the Glutamate (Glu) binding domain at the N terminus and the NAD binding doman—and a 48-residue antenna-like projection that extends from the top of each NAD binding domain, separated by a large active site cleft [5]. 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. Domain I is made up of residues 4-181 and 400-421, and is responsible for directing the assembly of the subunits into a hexamer. Domain I is colored blue. Domain II makes up the glutamate-binding domain, and is composed of mainly beta sheets involving residues 182-399, which is colored orange [6]. Domain I is also called the C-domain, whereas the Domain II is called the N-domain [7]. The NAD+ cofactor binds at the C-terminal end of the parallel beta strands in the N-domain, lying in the cleft between the N and C-domains. The glutamate substrate binds deep in this cleft, with the side chain of the glutamate lying in a pocket on the enzyme surface. Residues 193-204, and 383-393 are essential for the glutamate to bind in the cleft [8]. These residues are colored [9].


Figure 2. Each domain is colored differently - Glu-BD, NAD(P)-BD, antenna, the pivot helix. The allosteric regulators are shown as sphere models. This particular structure of GDH is a combination of two X-ray structures - one with a bound GTP (1HWZ) and the second one with a bound ADP (1NQT). Although not real, this structure shows the relative position of the allosteric effectors when bound to GDH. NADPH and Glu are shown as well.


File:Closed.jpg
Figure 3. When GDH is bound to Glutamate (blue) it's cleft is closed.

Located on top of the glutamate binding domain, these NAD+ binding domains rotate down upon the substrate and coenzyme to initiate catalysis. The forty eight-residue “antenna” that extends from the top of the NAD+ binding domain undergoes conformational changes as the cleft of the active site opens and closes [10]. When GDH is not bound by glutamate its cleft is open, however, when GDH is bound to glutamate it is closed. This position difference between the two domains allows the cleft to be closed, which brings the C4 of the nicotinamide ring and the alpha carbon of the glutamate substrate into the appropriate orientation for a hydride transfer to occur. Residues 200-206, 375-379, and 421-423 are critical for the control of the hinges that open or close the cleft between the two domains [11]. These residues are colored The residues that form this hinge, which allow the cleft to open or close are both near and far from the active site. The of GDH is composed of residues: 209-210, 213, 217, 261, 265, 289, 292, 450.

The N-terminal glutamate (Glu) binding domains, composed of mainly beta sheets,are mainly responsible in the build up of the core structure of the hexamer, a stacked dimer of trimers. The NAD+ binding domain and Glu binding domain form the catalytic cleft. During substrate binding, the NAD+ binding domain moves significantly. This movement has two components, rotating along the long axis of a helix at the back of the NAD+ binding domain, called "the pivot helix", and twisting about the antenna in a clockwise fashion. A comparison of the open and closed conformations of GDH reveals changes in the small helix of the descending strand of the antenna, which seems to recoil as the catalytic cleft opens [12].

The Glu binding domains of the monomers are position as 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.

Glutamate Dehydrogenase MechanismGlutamate Dehydrogenase Mechanism

Figure 4. Mechanism of Glutamate Dehydrogenase

The first step in the mechanism for catalytic activity of GDH is the deprotonation of the α-amino group of glutamate via Asp 165, which acts as a general base. Next, a hydride transfer to NAD+ occurs, forming a Schiff base intermediate [13]. During the first step a large movement between C-domain and N-domain occurs, which closes the cleft and brings C4 of the nicotinamide ring and the α-carbon of the substrate into the correct position for a hydride transfer [14]. The second step in the mechanism of GDH involves the attack of a water molecule on the Schiff base intermediate. This step is enhanced by Lys 125. The direction of the attack is very specific, so that the stereochemistry of the developing carbinolamine will be the L isomer and not the D isomer. During the generation of the carbinolamine intermediate and its conversion to alpha ketoglutarate, residue Asp 165 is very crucial for the transfer of the protons to and from the substrate. The final step that GDH catalyzes involves the loss of a single proton from each Lys 125 and Asp 165, which is transferred from water to GDH [15].



Regulation of Glutamate DehydrogenaseRegulation of Glutamate Dehydrogenase

GTP:

  • GTP is a potent inhibitor for the reaction and binds at the base of the antenna, wedged in between the NAD binding domain and the pivot helix. This binding site is only available for GTP binding when the catalytic cleft is closed. Therefore, after GTP binds to the 'closed' conformation it is more difficult for the 'mouth' to open and release either NAD+ and NADP+ as coenzyme [16].

GDP:

  • GDP is an activator.

ADP:

  • In the reductive amination reaction, ADP is a potent activator at low pH and low substrate concentration. At pH 6.0, high concentrations of α-ketoglutarate and NADH, inhibit the reaction. This substrate inhibition is alleviated by ADP. Therefore, while GTP and glutamate bind synergistically with NADH to inhibit GDH, ADP activates the reaction by decreasing the affinity of the active site. However, under conditions where the enzyme is not saturated (e.g. low substrate concentrations), this loss in binding affinity causes inhibition. [17].
  • Inhibition by high [ADP] is due to competition between ADP and the adenosine moiety of the coenzyme at the active site 1 [18].

NADH:

  • NADH, is another major allosteric inhibitor of GDH. [19].

NAD+:

  • This oxidized coenzyme binding causes activation. [20].

ATP: Different concentration levels of ATP have different effects on GDH activity:

  • Low [ATP] causes inhibition due to mediated through the GTP binding site. [21].
  • Intermediate [ATP] causes activation, mediated through the ADP effector site. [22].
  • High [ATP] concentration causes inhibition due to a competition between ATP and the adenosine moiety of the coenzyme at the active site [23].

ImplicatonsImplicatons

Figure 5. This schematic shows how the loss GTP inhibition can cause the hyperstimulated secretion of insulin (top) and the elevated serum levels of ammonium (bottom). In the pancreas, the loss of GTP inhibition increases the flux of glutamate to the Krebs cycle, leading to elevated ATP levels and secretion of insulin. In the liver, not only does accelerated catabolism increase the levels of ammonium, but the lower levels of glutamate also decrease the production of N-acetylglutamate.

Hyperosmolar hyperglycemic state (HHS) was one of the first diseases that clearly linked GDH regulation to insulin and ammonia homeostasis. Recent studies demonstrate that the activation of GDH was tightly correlated with increased glutaminolysis and release of insulin. HHS syndrome is caused by the loss of GTP regulation of GDH. Children with HHS have increased β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. This is due to uncontrolled catabolism of amino acids yielding high ATP levels that stimulate insulin secretion and high serum levels of ammonium. The elevation of serum ammonia levels induces an altered regulation of GDH, leading to increased ammonia production from glutamate oxidation. In addition to that, it can cause impaired urea synthesis by carbmoylphosphate synthetase (CPS) due to reduced formation of N-actyl-glutamate (activator) from glutamate (figure 4). This genetic lesion disrupts the regulator linkage between glycolysis and amino acid catabolism. [24].

ApplicationsApplications

GDH can be measured in a medical laboratory to evaluate the liver function. Elevated blood serum GDH levels indicate liver damage and GDH plays an important role in the differential diagnosis of liver disease, especially in combination with aminotransferases. GDH is localised in mitochondria, therefore practically none is liberated in generalised inflammatory diseases of the liver such as viral hepatitides. Liver diseases in which necrosis of hepatocytes is the predominant event, such as toxic liver damage or hypoxic liver disease, are characterised by high serum GDH levels. GDH is important for distinguishing between acute viral hepatitis and acute toxic liver necrosis or acute hypoxic liver disease, particularly in the case of liver damage with very high aminotransferases. In clinical trials, GDH can serve as a measurement for the safety of a drug.

IsozymesIsozymes

  • GLUD1
  • GLUD2

ReferencesReferences

  1. ↑ Stryer (Ed.). Biochemistry (5th Ed.) 2002. W.H. Freeman and Company, New York.
  2. ↑ Baker PJ, Waugh ML, Wang XG, Stillman TJ, Turnbull AP, Engel PC, Rice DW. Determinants of substrate specificity in the superfamily of amino acid dehydrogenases. Biochemistry. 1997 Dec 23;36(51):16109-15. PMID:9405044 doi:10.1021/bi972024x
  3. ↑ http://www.rcsb.org/pdb
  4. ↑ http://www.rcsb.org/pdb
  5. ↑ Nakasako M, Fujisawa T, Adachi S, Kudo T, Higuchi S. Large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase from Thermococcus profundus studied by cryogenic X-ray crystal structure analysis and small-angle X-ray scattering. Biochemistry. 2001 Mar 13;40(10):3069-79. PMID:11258921
  6. ↑ Goda S, Kojima M, Nishikawa Y, Kujo C, Kawakami R, Kuramitsu S, Sakuraba H, Hiragi Y, Ohshima T. Intersubunit interaction induced by subunit rearrangement is essential for the catalytic activity of the hyperthermophilic glutamate dehydrogenase from Pyrobaculum islandicum. Biochemistry. 2005 Nov 22;44(46):15304-13. PMID:16285734 doi:http://dx.doi.org/10.1021/bi050478l
  7. ↑ Nakasako M, Fujisawa T, Adachi S, Kudo T, Higuchi S. Large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase from Thermococcus profundus studied by cryogenic X-ray crystal structure analysis and small-angle X-ray scattering. Biochemistry. 2001 Mar 13;40(10):3069-79. PMID:11258921
  8. ↑ Baker PJ, Waugh ML, Wang XG, Stillman TJ, Turnbull AP, Engel PC, Rice DW. Determinants of substrate specificity in the superfamily of amino acid dehydrogenases. Biochemistry. 1997 Dec 23;36(51):16109-15. PMID:9405044 doi:10.1021/bi972024x
  9. ↑ Baker PJ, Waugh ML, Wang XG, Stillman TJ, Turnbull AP, Engel PC, Rice DW. Determinants of substrate specificity in the superfamily of amino acid dehydrogenases. Biochemistry. 1997 Dec 23;36(51):16109-15. PMID:9405044 doi:10.1021/bi972024x
  10. ↑ Banerjee S, Schmidt T, Fang J, Stanley CA, Smith TJ. Structural studies on ADP activation of mammalian glutamate dehydrogenase and the evolution of regulation. Biochemistry. 2003 Apr 1;42(12):3446-56. PMID:12653548 doi:http://dx.doi.org/10.1021/bi0206917
  11. ↑ Baker PJ, Waugh ML, Wang XG, Stillman TJ, Turnbull AP, Engel PC, Rice DW. Determinants of substrate specificity in the superfamily of amino acid dehydrogenases. Biochemistry. 1997 Dec 23;36(51):16109-15. PMID:9405044 doi:10.1021/bi972024x
  12. ↑ Smith TJ, Schmidt T, Fang J, Wu J, Siuzdak G, Stanley CA. The structure of apo human glutamate dehydrogenase details subunit communication and allostery. J Mol Biol. 2002 May 3;318(3):765-77. PMID:12054821 doi:10.1016/S0022-2836(02)00161-4
  13. ↑ Stillman TJ, Baker PJ, Britton KL, Rice DW. Conformational flexibility in glutamate dehydrogenase. Role of water in substrate recognition and catalysis. J Mol Biol. 1993 Dec 20;234(4):1131-9. PMID:8263917 doi:http://dx.doi.org/10.1006/jmbi.1993.1665
  14. ↑ Baker PJ, Waugh ML, Wang XG, Stillman TJ, Turnbull AP, Engel PC, Rice DW. Determinants of substrate specificity in the superfamily of amino acid dehydrogenases. Biochemistry. 1997 Dec 23;36(51):16109-15. PMID:9405044 doi:10.1021/bi972024x
  15. ↑ Stillman TJ, Baker PJ, Britton KL, Rice DW. Conformational flexibility in glutamate dehydrogenase. Role of water in substrate recognition and catalysis. J Mol Biol. 1993 Dec 20;234(4):1131-9. PMID:8263917 doi:http://dx.doi.org/10.1006/jmbi.1993.1665
  16. ↑ D'Mello, J. P. F.. "Glutamate Dehydrogenase." Amino Acids in Human Nutrition and Health. 2012. 1-23. Print
  17. ↑ D'Mello, J. P. F.. "Glutamate Dehydrogenase." Amino Acids in Human Nutrition and Health. 2012. 1-23. Print
  18. ↑ D'Mello, J. P. F.. "Glutamate Dehydrogenase." Amino Acids in Human Nutrition and Health. 2012. 1-23. Print
  19. ↑ D'Mello, J. P. F.. "Glutamate Dehydrogenase." Amino Acids in Human Nutrition and Health. 2012. 1-23. Print
  20. ↑ D'Mello, J. P. F.. "Glutamate Dehydrogenase." Amino Acids in Human Nutrition and Health. 2012. 1-23. Print
  21. ↑ Fang J, Hsu BY, MacMullen CM, Poncz M, Smith TJ, Stanley CA. Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations. Biochem J. 2002 Apr 1;363(Pt 1):81-7. PMID:11903050
  22. ↑ Fang J, Hsu BY, MacMullen CM, Poncz M, Smith TJ, Stanley CA. Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations. Biochem J. 2002 Apr 1;363(Pt 1):81-7. PMID:11903050
  23. ↑ Fang J, Hsu BY, MacMullen CM, Poncz M, Smith TJ, Stanley CA. Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations. Biochem J. 2002 Apr 1;363(Pt 1):81-7. PMID:11903050
  24. ↑ D'Mello, J. P. F.. "Glutamate Dehydrogenase." Amino Acids in Human Nutrition and Health. 2012. 1-23. Print

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