User:Wayne Chang
From Proteopedia
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University of Maryland, Baltimore County (UMBC). | University of Maryland, Baltimore County (UMBC). | ||
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| - | == Script Exercises == | ||
| - | <applet load='2qc8' size='300' frame='true' align='right' /> | ||
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| - | <scene name='User:Wayne_Chang/Glutamate_synthase/1'>Exercise 1: Backbone Trace with ligand</scene> | ||
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| - | Exercise shows a backbone trace of Glutamate Synthase which allows the ligands inside ADP, P3S, Cl- and Mn2+ to be seen. | ||
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| - | <scene name='User:Wayne_Chang/Glutamate_synthase/2'>Exercise 2: Ligand and Chain Selection with Labeling</scene> | ||
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| - | Isolates chain A of Glutamate Synthase and labels the ligands for easy identification. | ||
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| - | <scene name='User:Wayne_Chang/Glutamate_synthase/3'>Exercise 3: Active Site Residues</scene> | ||
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| - | Wire Structure of Active Site residues of chain A using information obtained from PDBsum entry for Glutamate Synthase. | ||
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| - | <scene name='User:Wayne_Chang/Glutamate_synthase/4'>Exercise 4: Going Solo</scene> | ||
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| - | Still picture of salt bridge between residue 63 of chain F and residue 319 of chain G. Bridge length is also provided in Angstroms. | ||
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| - | == Outline == | ||
| - | <applet load='2qc8' size='300' frame='true' align='right' /> | ||
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| - | '''-- Work in Progress -- ''' | ||
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| - | Glutamine synthetase (GS) catalyzes the ATP dependent condensation of glutamate and ammonia, producing, glutamine, ADP, and an inorganic phosphate group. | ||
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| - | Glutamate + ATP + NH3 → Glutamine + ADP + phosphate | ||
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| - | Ammonium ion is thought to bind to GS at the monovalent cation binding site for Tl(+) and Cs(+) ions. | ||
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| + | <applet load='1lgr' size='300' frame='true' align='right' /> | ||
| + | Glutamine Synthetase (GS) catalyzes the ATP dependent condensation of glutamate and ammonia. The reaction produces glutamine, ADP, and an inorganic phosphate group<ref> Liaw, S-H, et.al.,Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site Protein Sci. 1995 4: 2358-2365[http://www.proteinscience.org/cgi/content/abstract/4/11/2358].</ref>: | ||
| + | :::::::Glutamate + ATP + NH<sub>4</sub><sup>+</sup> → Glutamine + ADP + Pi | ||
| + | '''Active Site Structure''' | ||
| + | Bacterial GS consists of <scene name='User:Wayne_Chang/All_ammonium_binding_sites/1'>12 identical binding sites</scene> with 622 symmetry. There are 12 funnel shaped active sites located at the interface of each subunit. The <scene name='User:Wayne_Chang/Funnel_active_site/4'>funnel</scene> is open at the top as well as at the bottom and is about 30 Å <scene name='User:Wayne_Chang/Funnel_active_site_top/2'>wide at its top</scene>, about 10 Å <scene name='User:Wayne_Chang/Funnel_active_site_bottom/2'>wide at the bottom</scene>. Each funnel consists of 4 binding sites for the ligands ATP, Mn, Glutamate, and Ammonium ion all of which participate in the reaction GS catalyzes (color coding of binding sites in the funnel: <span style="color:green">Glutamate</span>, <span style="color:blue">ATP</span>, <span style="color:purple">Mn<sup>2+</sup></span>,<span style="color:red"> NH<sub>4</sub><sup>+</sup></span>). ATP binds at the top of the active site, while glutamate binds at the bottom. GS is a trimetallic enzyme, binding two divalent ions (Mn<sup>2+</sup> or Mg<sup>2+</sup>) and one monovalent ion (NH<sub>4</sub><sup>+</sup>).<sup>[1]</sup> | ||
| + | '''Ammonium binding site - a Negatively charged pocket''' | ||
| + | The four residues that create the ammonium binding site are <scene name='User:Wayne_Chang/Ammonium_binding_site/2'>Glu 212, Tyr 179, Ser 53', and Asp 50'</scene>. Residues Serine 53 and Asp 50 are found on the adjacent chain which explains the apostrophe after their numbers. All four are negatively charged which is important for the ammonium ion to bind favorably with GS and attack y-glutamyl phosphate. If a water molecule instead of an ammonium ion were to attack y-glutamyl phosphate, the product would be glutamate instead of glutamine. In addition, a negatively charged pocket serves to provide stability with the positive ammonium ion in the active conformation. The proximity of the residues to the glutamate binding site as well as the affinity for the positive ammonium molecule to the negative residues helps to catalyze the reaction.<sup>[1]</sup> | ||
| - | + | '''Catalysis''' | |
| - | + | The reaction is catalyzed in a mulit-step process. Available evidence shows that ammonium ion, and not ammonia, is the initial substrate in the reaction. A deprotonated ammonia completes the reaction after an ammonium ion binds to the negatively charged pocket of GS. In the first step, glutamate reacts with ATP to produce ADP and the intermediate y-glutamyl phosphate. ADP binds to Asp 50’. The ADP binding induces Asp 50’ to move toward the ammonium binding site to enhance ammonium binding. Ammonium is then deprotonated, perhaps by Asp 50’, to form ammonia that reacts with the intermediate y-glutamyl phosphate, yielding glutamine. <sup>[1]</sup><ref> Liaw SH, Eisenberg D. Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes. Biochemistry. 1994 Jan 25;33(3):675-81.</ref> | |
| - | + | ==References== | |
| + | <references/> | ||
Current revision
Assignment 12: IVC: Ammonium Binding Site
Mapping the Ammonium binding site and explaining how it contributes to catalysis.
Chang, Yu-Wei and Kaushal, Pankaj.
BIOL 430: Biological Chemistry.
University of Maryland, Baltimore County (UMBC).
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Glutamine Synthetase (GS) catalyzes the ATP dependent condensation of glutamate and ammonia. The reaction produces glutamine, ADP, and an inorganic phosphate group[1]:
- Glutamate + ATP + NH4+ → Glutamine + ADP + Pi
Active Site Structure
Bacterial GS consists of with 622 symmetry. There are 12 funnel shaped active sites located at the interface of each subunit. The is open at the top as well as at the bottom and is about 30 Å , about 10 Å . Each funnel consists of 4 binding sites for the ligands ATP, Mn, Glutamate, and Ammonium ion all of which participate in the reaction GS catalyzes (color coding of binding sites in the funnel: Glutamate, ATP, Mn2+, NH4+). ATP binds at the top of the active site, while glutamate binds at the bottom. GS is a trimetallic enzyme, binding two divalent ions (Mn2+ or Mg2+) and one monovalent ion (NH4+).[1]
Ammonium binding site - a Negatively charged pocket
The four residues that create the ammonium binding site are . Residues Serine 53 and Asp 50 are found on the adjacent chain which explains the apostrophe after their numbers. All four are negatively charged which is important for the ammonium ion to bind favorably with GS and attack y-glutamyl phosphate. If a water molecule instead of an ammonium ion were to attack y-glutamyl phosphate, the product would be glutamate instead of glutamine. In addition, a negatively charged pocket serves to provide stability with the positive ammonium ion in the active conformation. The proximity of the residues to the glutamate binding site as well as the affinity for the positive ammonium molecule to the negative residues helps to catalyze the reaction.[1]
Catalysis
The reaction is catalyzed in a mulit-step process. Available evidence shows that ammonium ion, and not ammonia, is the initial substrate in the reaction. A deprotonated ammonia completes the reaction after an ammonium ion binds to the negatively charged pocket of GS. In the first step, glutamate reacts with ATP to produce ADP and the intermediate y-glutamyl phosphate. ADP binds to Asp 50’. The ADP binding induces Asp 50’ to move toward the ammonium binding site to enhance ammonium binding. Ammonium is then deprotonated, perhaps by Asp 50’, to form ammonia that reacts with the intermediate y-glutamyl phosphate, yielding glutamine. [1][2]
References
- ↑ Liaw, S-H, et.al.,Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site Protein Sci. 1995 4: 2358-2365[1].
- ↑ Liaw SH, Eisenberg D. Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes. Biochemistry. 1994 Jan 25;33(3):675-81.
