Sandbox Reserved 761: Difference between revisions

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[[Image:traces.jpg|frame|left|Figure 1. Image of Glutamate Dehydrogenase]]

'''Glutamate Dehydrogenase''' (GDH) is a homohexameric enzyme that catalyses the reversible oxidative deamination of L-glutamate to α-ketoglutarate, and vice versa using NAD+ and/or NADP+ as ~~coenzymes~~. Located in the mitochondria, GDH is an ~~important branch-point~~ enzyme ~~between carbon and nitrogen metabolism~~.

'''Glutamate Dehydrogenase''' (GDH) is a homohexameric enzyme 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 nitrogen and glutamate (Glu) metabolism and the energy homeostasis. GDH is expressed at high levels in liver, brain, pancreas and kidney, but not in muscle. In the pancreatic cells, GDH is thought to be involved in insulin secretion mechanisms. In nervous tissue, where Glu is present in concentrations higher than in the other tissues, GDH appears to function in both the synthesis and the catabolism of Glu and in ammonia detoxification. A dehydrogenase is an enzyme that oxidizes a substrate by a reduction reaction that transfers one or more hydrides (H−) to an electron acceptor, usually NAD+/NADP+. 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+ <ref>PMID:NBK21154</ref>. 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 <ref>PMID:9405044</ref>. 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.

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+ <ref>PMID:NBK21154</ref>. 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 <ref>PMID:9405044</ref>. 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 Structure==

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==Glutamate Dehydrogenase Mechanism==

The first step in the mechanism for catalytic activity of GDH is the -deprotonation of the alpha-amino group of glutamate by Asp 165, which acts as a general base. Next, a hydride transfer to NAD+ occurs, which forms 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 alpha carbon of the substrate into the correct position for a hydride transfer (1). The second step involves the attack of a water molecule on the Schiff base intermediate, which 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 (13).

The first step in the mechanism for catalytic activity of GDH is the -deprotonation of the alpha-amino group of glutamate by Asp 165, which acts as a general base. Next, a hydride transfer to NAD+ occurs, which forms a Schiff base intermediate <ref>PMID:8263917</ref>. 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 alpha carbon of the substrate into the correct position for a hydride transfer (1). The second step involves the attack of a water molecule on the Schiff base intermediate, which 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 (13).

[[Image:GDH.jpg|frame|left|Figure 1. Mechanism of Glutamate Dehydrogenase]]

==Regulation 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 (1).

'''ADP'''

In the reductive amination reaction, ADP is a potent activator at low pH and low substrate concentration. ADP 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 GDH hexamers form long polymers in the crystal cell with more interactions than found in the abortive complex crystals. However, at pH 6.0 and high concentrations of α-ketoglutarate and NADH, the reaction is inhibited. 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.

'''NADH'''

NADH, is another major allosteric inhibitor of GDH

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 ~~(1).~~

'''NAD+'''

This oxidized coenzyme binding causes activation

In the ~~reductive amination reaction, ADP is a potent activator at low pH and low substrate concentration~~. ~~However, at pH 6.0 and high concentrations of α~~-~~ketoglutarate and NADH~~, the ~~reaction is inhibited. 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~~.

'''ATP'''

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

-Low [ATP] causes inhibition due to mediated through the GTP binding site.

-Intermediate [ATP] causes activation, mediated through the ADP effector site

-High [ATP] concentration causes inhibition due to a competition between ATP and the adenosine moiety of the coenzyme at the active site <ref>PMID:11903050</ref>.

==Implicatons==

==References==

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@@ Line 11: / Line 11: @@
 [[Image:traces.jpg|frame|left|Figure 1. Image of Glutamate Dehydrogenase]]
-'''Glutamate Dehydrogenase''' (GDH) is a homohexameric enzyme that catalyses the reversible oxidative deamination of L-glutamate to α-ketoglutarate, and vice versa using NAD+ and/or NADP+ as coenzymes. Located in the mitochondria, GDH is an important branch-point enzyme between carbon and nitrogen metabolism.
+'''Glutamate Dehydrogenase''' (GDH) is a homohexameric enzyme 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 nitrogen and glutamate (Glu) metabolism and the energy homeostasis. GDH is expressed at high levels in liver, brain, pancreas and kidney, but not in muscle. In the pancreatic cells, GDH is thought to be involved in insulin secretion mechanisms. In nervous tissue, where Glu is present in concentrations higher than in the other tissues, GDH appears to function in both the synthesis and the catabolism of Glu and in ammonia detoxification. A dehydrogenase is an enzyme that oxidizes a substrate by a reduction reaction that transfers one or more hydrides (H−) to an electron acceptor, usually NAD+/NADP+. 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+ <ref>PMID:NBK21154</ref>.  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 <ref>PMID:9405044</ref>.  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.
-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+ <ref>PMID:NBK21154</ref>.  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 <ref>PMID:9405044</ref>.  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 Structure==
@@ Line 35: / Line 35: @@
 ==Glutamate Dehydrogenase Mechanism==
-The first step in the mechanism for catalytic activity of GDH is the -deprotonation of the alpha-amino group of glutamate by Asp 165, which acts as a general base.  Next, a hydride transfer to NAD+ occurs, which forms 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 alpha carbon of the substrate into the correct position for a hydride transfer (1).  The second step involves the attack of a water molecule on the Schiff base intermediate, which 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 (13).
+The first step in the mechanism for catalytic activity of GDH is the -deprotonation of the alpha-amino group of glutamate by Asp 165, which acts as a general base.  Next, a hydride transfer to NAD+ occurs, which forms a Schiff base intermediate <ref>PMID:8263917</ref>.  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 alpha carbon of the substrate into the correct position for a hydride transfer (1).  The second step involves the attack of a water molecule on the Schiff base intermediate, which 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 (13).
 [[Image:GDH.jpg|frame|left|Figure 1. Mechanism of Glutamate Dehydrogenase]]
+==Regulation 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 (1).
+'''ADP'''
+In the reductive amination reaction, ADP is a potent activator at low pH and low substrate concentration. ADP 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 GDH hexamers form long polymers in the crystal cell with more interactions than found in the abortive complex crystals. However, at pH 6.0 and high concentrations of α-ketoglutarate and NADH, the reaction is inhibited. 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.
+'''NADH'''
+NADH, is another major allosteric inhibitor of GDH
-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 (1).
+'''NAD+'''
+This oxidized coenzyme binding causes activation
-In the reductive amination reaction, ADP is a potent activator at low pH and low substrate concentration. However, at pH 6.0 and high concentrations of α-ketoglutarate and NADH, the reaction is inhibited. 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.
+'''ATP'''
+Different concentration levels of ATP have different effects on GDH activity:
+-Low [ATP] causes inhibition due to mediated through the GTP binding site.
+-Intermediate [ATP] causes activation, mediated through the ADP effector site
+-High [ATP] concentration causes inhibition due to a competition between ATP and the adenosine moiety of the coenzyme at the active site <ref>PMID:11903050</ref>.
 ==Implicatons==
 ==References==