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Glutamate Dehydrogenase (GluDH) is a member of the superfamily of amino acid dehydrogenase and functions in the cell to dehydrate α-ketoglutarate to the amino acid glutamate and also to perform the reverse reaction.<ref name="1bgv">PMID:8263917</ref>  
Glutamate Dehydrogenase (GluDH) is a member of the superfamily of amino acid dehydrogenase and functions in the cell to dehydrate α-ketoglutarate to the amino acid glutamate and also to perform the reverse reaction.<ref name="1bgv">PMID:8263917</ref>  
GluDH is at the threshold of carbon metabolism (GluDH feeds α-ketoglutarate into the tricarboxylic acid cycle) and nitrogen metabolism (the amine product is utilized by other biosynthetic pathways).<ref name="1hwxyz">PMID:11254391</ref>. Due to its prominent position on the threshold between catabolic and anabolic pathways, GluDH is ubiquitously expressed in both complex and simple organisms.<ref name="1hwx">PMID:10425679</ref>


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  Image:Alpha-ketoglutamate2.png |[[α-Ketoglutaraic acid]]
  Image:Alpha-ketoglutamate2.png |[[α-Ketoglutaraic acid]]
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GluDH is at the threshold of carbon metabolism (GluDH feeds α-ketoglutarate into the tricarboxylic acid cycle) and nitrogen metabolism (the amine product is utilized by other biosynthetic pathways).<ref name="1hwxyz">PMID:11254391</ref>. Due to its prominent position on the threshold between catabolic and anabolic pathways, GluDH is ubiquitously expressed in both complex and simple organisms.<ref name="1hwx">PMID:10425679</ref>
In vertebrates and plants, GluDH is preferentially found in the mitochondria, but also in the cytoplasm. In prokaryotes it is found in the cytosol.<ref>[http://www.brenda-enzymes.org/php/flat_result.php4?ecno=1.4.1.2&organism_list=&Suchword=&UniProtAcc=#LOCALIZATION EC 1.4.1.2] Brenda 2010</ref>


Reductive amination of α-ketoglutarate (α-KG) is the process by which the ketone is converted to an amine via an imine intermediate. The reverse reaction, oxidative deamination, is the conversion of the amine functional group to a ketone.
Reductive amination of α-ketoglutarate (α-KG) is the process by which the ketone is converted to an amine via an imine intermediate. The reverse reaction, oxidative deamination, is the conversion of the amine functional group to a ketone.
In vertebrates the produced ammonia is usually utilized in the urea cycle and in bacteria the ammonia is assimilated to amino acids and amidotransferases.<ref name="Lightfoot_1988">Lightfoot DA, Baron AJ, Wootton JC (1988). "Expression of the Escherichia coli glutamate dehydrogenase gene in the cyanobacterium Synechococcus PCC6301 causes ammonium tolerance". Plant Molecular Biology 11 (3): 335-344. [http://www.springerlink.com/content/w2721u62r8021510/ doi 10.1007/BF00027390]</ref>


Glutamate dehydrogenase shares sequence homology and structural homology to the superfamily of amino acid dehydrogenases, which supports the idea that this superfamily formed by divergent evolution. <ref name="1bgv" /> Because of the homology among all proteins in this superfamily, many dehydrogenases can work on multiple substrates. Nonetheless, GluDH appears to be very specific towards its substrates.
Glutamate dehydrogenase shares sequence homology and structural homology to the superfamily of amino acid dehydrogenases, which supports the idea that this superfamily formed by divergent evolution. <ref name="1bgv" /> Because of the homology among all proteins in this superfamily, many dehydrogenases can work on multiple substrates. Nonetheless, GluDH appears to be very specific towards its substrates.
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Like most prokaryotic GluDH, Mammalian GluDH has been found to hexamerize as a dimer of  
Like most prokaryotic GluDH, Mammalian GluDH has been found to hexamerize as a dimer of  
<scene name='User:Cameron_Evans/Sandbox_1/Human_d_e_f/1'>trimers</scene>.  
<scene name='User:Cameron_Evans/Sandbox_1/Human_d_e_f/4'>trimers</scene>.  


Furthermore,each <scene name='User:Cameron_Evans/Sandbox_1/Human_d_alone/1'>monomer</scene> of mammalian GluDH, like prokaryotic GluDH, is composed of two domains: Domain I, which is responsible for the assembly of the hexamer; and Domain II, which is responsible for dinucleotide binding (both true statements for prokaryotic GluDH). Furthermore, each domain is similar to the domains within csGluDH as <scene name='User:Cameron_Evans/Sandbox_1/Bogdh_2ndary/1'>each is a beta sheet flanked by alpha helices</scene>.
Furthermore,each <scene name='User:Cameron_Evans/Sandbox_1/Human_d_alone/1'>monomer</scene> of mammalian GluDH, like prokaryotic GluDH, is composed of two domains: Domain I, which is responsible for the assembly of the hexamer; and Domain II, which is responsible for dinucleotide binding (both true statements for prokaryotic GluDH). Furthermore, each domain is similar to the domains within csGluDH as <scene name='User:Cameron_Evans/Sandbox_1/Bogdh_2ndary/1'>each is a beta sheet flanked by alpha helices</scene>.


Unlike csGluDH, the boGluDH monomer has 48 residue <scene name='User:Cameron_Evans/Sandbox_1/Tail_1/1'>"antenna"</scene> that assists in the trimerization process. <scene name='User:Cameron_Evans/Sandbox_1/Human_d_e_f/2'>(The interactions of the antennae are best seen in the trimer)</scene>. These antennae appear to undergo conformational changes as the "mouth" of GluDH opens and closes.
Unlike csGluDH, the boGluDH monomer has 48 residue <scene name='User:Cameron_Evans/Sandbox_1/Tail_1/1'>"antenna"</scene> that assists in the trimerization process. <scene name='User:Cameron_Evans/Sandbox_1/Human_d_e_f/6'>(The interactions of the antennae are best seen in the trimer)</scene>. These antennae appear to undergo conformational changes as the "mouth" of GluDH opens and closes.


This 48 residue insertion (397-444), which does not exist in the sequences of prokaryotic GluDH, is thought to be significant for the allosteric interactions that distinguish mammalian GluDH from prokaryotic GluDH (see specific interactions below).<ref name=1hwx />
This 48 residue insertion (397-444), which does not exist in the sequences of prokaryotic GluDH, is thought to be significant for the allosteric interactions that distinguish mammalian GluDH from prokaryotic GluDH (see specific interactions below).<ref name=1hwx />
The location of the clefts around the hexamer are more evident in <scene name='User:Cameron_Evans/Sandbox_1/2_hex/1'>this scene,</scene> which  x illustrates the secondary structure of one monomer within the hexamer


===Specificity===
===Specificity===
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The  
The  
<scene name='User:Cameron_Evans/Sandbox_1/Gtp_n_helix/1'>GTP binds </scene> between the active site of the monomer and the <color=red> pivot helix </color>. It is thought that GTP causes negative regulation of GluDH by increasing the enzyme's affinity for the product to the extent that the release of the product is the rate limiting step of the overall reaction.
<scene name='User:Cameron_Evans/Sandbox_1/Gtp_n_helix/1'>GTP binds </scene> between the active site of the monomer and the <font color='red'>pivot helix </font>. It is thought that GTP causes negative regulation of GluDH by increasing the enzyme's affinity for the product to the extent that the release of the product is the rate limiting step of the overall reaction.


When the enzyme is highly saturated, the enzyme has been found to form an "abortive complex" that is the cofactor and the reagent locked in a non-catalytic conformation. Upon the binding of a positive regulator, like GDP or ADP, the protein is thought to be forced into the open conformation, driving the reaction is allowed to completion. ADP has also been shown to decrease the affinity of the enzyme to its products.
When the enzyme is highly saturated, the enzyme has been found to form an "abortive complex" that is the cofactor and the reagent locked in a non-catalytic conformation. Upon the binding of a positive regulator, like GDP or ADP, the protein is thought to be forced into the open conformation, driving the reaction is allowed to completion. ADP has also been shown to decrease the affinity of the enzyme to its products.
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=====Dinucleotides=====
=====Dinucleotides=====
The NAD(P)(H) coenzyme is has also been shown to <scene name='User:Cameron_Evans/Sandbox_1/2nd_cofactor/3'>bind in the same general region the other allosteric regulators bind</scene> - that is, between the pivot helix (RED) and the active site. Furthermore, this binding site appears to bind NAD(H) with ten times the affinity as NADP(H). As might be expected, the binding of the reduced coenzyme inhibits oxidative deamination, and the binding of the oxidized form promotes deamination.  
The NAD(P)(H) coenzyme is has also been shown to <scene name='User:Cameron_Evans/Sandbox_1/2nd_cofactor/3'>bind in the same general region the other allosteric regulators bind</scene> - that is, between the <font color='red'>pivot helix </font> and the active site. Furthermore, this binding site appears to bind NAD(H) with ten times the affinity as NADP(H). As might be expected, the binding of the reduced coenzyme inhibits oxidative deamination, and the binding of the oxidized form promotes deamination.  


During reductive amination, NADPH is an inhibitor at pH 8, but not at pH7. Furthermore, NADPH binding is greatly enhanced in the presence of glutamate, where it inhibits the enzyme; however, the enhanced binding observed in the presence of ketoglutarate is not coupled with inhibition.
During reductive amination, NADPH is an inhibitor at pH 8, but not at pH7. Furthermore, NADPH binding is greatly enhanced in the presence of glutamate, where it inhibits the enzyme; however, the enhanced binding observed in the presence of ketoglutarate is not coupled with inhibition.
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In the apo form of csGluDH, K113 (domain I) is hydrogen bound to N373 (domain II) and stabilizes the open structure. On substrate binding, K113 moves to hydrogen bond to the alpha carbonyl of the substrate while maintaining contact with N373. This causes the conformational change which closes the cleft. Based on the crystal structures of csGluDH and previous binding studies with boGluDH, Stillman and Baker, ‘’et al’’ (1993) have proposed the following catalytic mechanism.
In the apo form of csGluDH, K113 (domain I) is hydrogen bound to N373 (domain II) and stabilizes the open structure. On substrate binding, K113 moves to hydrogen bond to the alpha carbonyl of the substrate while maintaining contact with N373. This causes the conformational change which closes the cleft. Based on the crystal structures of csGluDH and previous binding studies with boGluDH, Stillman and Baker, ‘’et al’’ (1993) have proposed the following catalytic mechanism.


After substrate binding and monomer closing, (1) the alpha amino of the glutamate is deprotonated by E165, and (2) hydride transfer to the ‘’Si’’ face of the coenzyme. (3) The change in substrate geometry is sensed by K133and the closed conformation is thought to be brought even closer together to facilitate hydride transfer. (4) Water attacks the iminoketoglutarate intermediate and (5) the protons gained by K125 and D165 in catalysis are lost and the monomer returns to the open conformation. (The Principle of Microreversability dictates that the mechanism for reductive amination can be easily described in a backwards fashion). <ref name="1bgv" />
After substrate binding and monomer closing, (1) the alpha amino of the glutamate is deprotonated by E165, and (2) hydride transfer to the ‘’Si’’ face of the coenzyme. (3) The change in substrate geometry is sensed by K133and the closed conformation is thought to be brought even closer together to facilitate hydride transfer. (4) Water attacks the iminoketoglutarate intermediate (to become the carbonyl oxygen) and (5) the protons gained by K125 and D165 in catalysis are lost and the monomer returns to the open conformation. (The Principle of Microreversability dictates that the mechanism for reductive amination can be easily described in a backwards fashion). <ref name="1bgv" />
 
The details of how ammonia fits into this mechanism remain unknown.<ref name="Lightfoot_1988" />


==References==
==References==
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<references />
<references />
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