User:Grace Natalie

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<font size=8 face="Comic Sans MS">The Glutamine Synthetase Project</font>
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<center><font size=6 face="Comic Sans MS">ATP Binding in Glutamine Synthetase</font></center>
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<br><br>
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<font size=4 face ="Arial">Background</font>
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<br>
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<applet load='1lgr' size='350' color='white' frame='true' align='right' caption='Glutamine Synthetase from Salmonella typhimurium'/>
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<font size=2>Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and
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glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations
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<ref name="Liaw">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</ref> .
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The reaction occurs in two steps with γ-glutamyl phosphate as an intermediate and is used by
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bacteria to introduce reduced nitrogen into cellular metabolism. GS is a dodecamer formed from
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two face-to-face hexameric rings of subunits, with 12 active sites formed between monomers
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<ref name="Book">Gill, H & Eisenberg, D., Biochemistry 2001 40: 1903-1912</ref> .</font>
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<br><br>
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<center><font size=4 face ="Arial">Overall Reaction of Glutamine Synthetase</font></center>
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<center><font size=2>Glutamate + NH<SUB>4</SUB><SUP>+</SUP> + ATP --> glutamine + ADP + P<SUB>i</SUB></font></center>
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<br>
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<font size=4 face ="Arial">Overall Mechanism</font>
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<br>
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<font size=2>The first step is the formation of the activated intermediate γ-glutamyl phosphate.
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The n2 ion coordinates the phosphate oxygens of ATP to allow phosphoryl transfer to the
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γ-carboxylate group of glutatmate, yielding the intermediate <ref name="Eisenberg">D. Eisneberg et al / Biochimica et Biophysica Acta 1477 (2000) 124</ref> . The second step is the attack on the intermediate by ammonia
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therefore releasing free phosphate to yield glutamine.</font>
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<br>
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<font size=4 face ="Arial">ATP binding site</font>
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<br>
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<font size=2>Each active site of GS is described as a 'bifunnel in which ATP and glutamate bind at opposite ends.
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The ATP binding site is referred to as the top of the bifunnel because it opens to the external 6-fold surface of GS (figure below) <ref name="Eisenberg"/>. At the the joint of the <scene name='User:Grace_Natalie/Atp_binding_site/2'> bifunnel </scene> are two cation binding sites, n1 and n2, where either magnesium or manganese bind
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for catalysis. The n2 ion is involved in the phosphroyl transfer, while the n1 ion stabilizes an active GS and plays a role in binding glutamate <ref name="Eisenberg"/> .
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<br>
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<font size=4 face ="Arial">Involving Residues</font><br>
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<font size=2>Most residues involved in enzymatic catalysis are located at the C domain but Asp50 is
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contributed from the N domain of the other subunit <scene name='User:Grace_Natalie/Involving_residues_at_one_site/1'>(View of involving residues)</scene> . Both the N-terminus and C-terminus of each subunit are helical.
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The N-terminal helix sits above the hexameric ring and is exposed to solvent <ref name="Eisenberg"/> .
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The C-terminal hexlix (helical thong) is inserted into the hydrophobic hole in the subunit opposite hexameric ring
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<ref name="Eisenberg"/> . The movement of Asp-50 aids in the formation of the ammonium binding site, and the movement of Arg-339 assist phosphoryl transfer and P<SUB>i</SUB> binding. <scene name='User:Grace_Natalie/Gln_synthetase_showing_asp50/1'>(Asp50 residue)</scene>.</font>
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<center>[[Image:Untitled.JPG]]<br>
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<font size=2>D. Eisneberg et al / Biochimica et Biophysica Acta 1477 (2000) 124</font></center>
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<br><br>
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<center><font size=4 face ="Arial">More Catalytic Residues</font><ref name="Liaw"/>
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<table border=1>
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<tr>
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<td width=60><B>Residue</B></td>
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<td width=370><B>Role in enzymatic mechanism</B></td>
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</tr>
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<tr>
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<td width=60>Arg-321</td>
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<td width=370>Coordinates the carboxylate of glutamate</td>
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</tr><tr>
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<td width=60>Glu-327</td>
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<td width=370>Closes active site and shields intermediate from hydrolysis</td>
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</tr>
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<tr>
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<td width=60>His-269</td>
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<td width=370>Coordinates the n2 ion</td>
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</tr><tr>
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<td width=60>Glu-220</td>
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<td width=370>Coordinates the n1 ion</td>
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</tr>
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<tr>
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<td width=60>Asp-50</td>
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<td width=370>Increases the affinity for ammonium binding</td>
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</tr>
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</table>
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<br>
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</center>
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<br><br>
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<font size=4 face ="Arial">References</font>
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<br>
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<font size=2> <references/><br>
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<br><br><br>
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University of Maryland Baltimore County - BioChemistry
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<applet load='2qc8' size='400' color='white' frame='true' align='right' caption='testcaption' />
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<scene name='User:Grace_Natalie/Backbone_trace_with_ligand/1'>Backbone trace with ligand of the Crystal structure of human glutamine synthetase</scene>
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<scene name='User:Grace_Natalie/Backbone_trace_with_ligand/3'>Ligand and Chain Selection With Labeling</scene>
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<scene name='User:Grace_Natalie/Active_site_amino_acid_residue/2'>Active Site Amino Acid Residues with Ligands</scene>
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<scene name='User:Grace_Natalie/Salt_bridge/1'>A salt bridge between Chain F Residue 63 and Chain G Residue 319 on a structure of human glutamine synthetase</scene>
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Current revision

ATP Binding in Glutamine Synthetase



Background

Glutamine Synthetase from Salmonella typhimurium

Drag the structure with the mouse to rotate

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations [1] . The reaction occurs in two steps with γ-glutamyl phosphate as an intermediate and is used by bacteria to introduce reduced nitrogen into cellular metabolism. GS is a dodecamer formed from two face-to-face hexameric rings of subunits, with 12 active sites formed between monomers [2] .

Overall Reaction of Glutamine Synthetase
Glutamate + NH4+ + ATP --> glutamine + ADP + Pi


Overall Mechanism
The first step is the formation of the activated intermediate γ-glutamyl phosphate. The n2 ion coordinates the phosphate oxygens of ATP to allow phosphoryl transfer to the γ-carboxylate group of glutatmate, yielding the intermediate [3] . The second step is the attack on the intermediate by ammonia therefore releasing free phosphate to yield glutamine.
ATP binding site
Each active site of GS is described as a 'bifunnel in which ATP and glutamate bind at opposite ends. The ATP binding site is referred to as the top of the bifunnel because it opens to the external 6-fold surface of GS (figure below) [3]. At the the joint of the are two cation binding sites, n1 and n2, where either magnesium or manganese bind for catalysis. The n2 ion is involved in the phosphroyl transfer, while the n1 ion stabilizes an active GS and plays a role in binding glutamate [3] .
Involving Residues
Most residues involved in enzymatic catalysis are located at the C domain but Asp50 is contributed from the N domain of the other subunit . Both the N-terminus and C-terminus of each subunit are helical. The N-terminal helix sits above the hexameric ring and is exposed to solvent [3] . The C-terminal hexlix (helical thong) is inserted into the hydrophobic hole in the subunit opposite hexameric ring [3] . The movement of Asp-50 aids in the formation of the ammonium binding site, and the movement of Arg-339 assist phosphoryl transfer and Pi binding. .

Image:Untitled.JPG
D. Eisneberg et al / Biochimica et Biophysica Acta 1477 (2000) 124



More Catalytic Residues[1]
Residue Role in enzymatic mechanism
Arg-321 Coordinates the carboxylate of glutamate
Glu-327 Closes active site and shields intermediate from hydrolysis
His-269 Coordinates the n2 ion
Glu-220 Coordinates the n1 ion
Asp-50 Increases the affinity for ammonium binding




References

  1. ↑ 1.0 1.1 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
  2. ↑ Gill, H & Eisenberg, D., Biochemistry 2001 40: 1903-1912
  3. ↑ 3.0 3.1 3.2 3.3 3.4 D. Eisneberg et al / Biochimica et Biophysica Acta 1477 (2000) 124

Proteopedia Page Contributors and Editors (what is this?)

Grace Natalie, Eran Hodis

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