User:Grace Natalie

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<font size=4 face ="Arial">Background</font>
<font size=4 face ="Arial">Background</font>
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<applet load='1lgr' size='350' color='white' frame='true' align='right' caption='Glutamine Synthetase from Salmonella typhimurium'/>
<font size=2>Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and
<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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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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<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> .
The reaction occurs in two steps with γ-glutamyl phosphate as an intermediate and is used by
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
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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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>
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<ref name="Book">Gill, H & Eisenberg, D., Biochemistry 2001 40: 1903-1912</ref> .</font>
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Each active site is described as a bifunnel allowing ATP and glutamate to bind at opposite ends.
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<ref name="Eisenberg">Eisenberg, D., et.al., Structure-function relationships of glutamine synthetases, Biochim Biophys Acta 2000: 1477, 122-145.</ref>
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Divalent metal ions n1 and n2 (Mg<SUP>2+</SUP> or Mn<SUP>2+</SUP>) bind at the center of the bifunnel
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and are important for catalysis and stutructural stability.</font>
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<br><br>
<br><br>
<center><font size=4 face ="Arial">Overall Reaction of Glutamine Synthetase</font></center>
<center><font size=4 face ="Arial">Overall Reaction of Glutamine Synthetase</font></center>
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<font size=2>The first step is the formation of the activated intermediate γ-glutamyl phosphate.
<font size=2>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
The n2 ion coordinates the phosphate oxygens of ATP to allow phosphoryl transfer to the
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γ-carboxylate group of glutatmate, yeilding the intermediate. <ref name="Eisenberg"/> The second step is 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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attack on the intermediate by ammonia therefore releasing free phosphate to yield glutamine.</font>
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therefore releasing free phosphate to yield glutamine.</font>
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<font size=4 face ="Arial">ATP binding site</font>
<font size=4 face ="Arial">ATP binding site</font>
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<br>
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<font size=2>ATP binds at the top of the active site cavity and the glutamate binds at the bottom,
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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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adjacent to the n1 ion <ref name="Liaw"/>. The movement of Arg 359 toward the
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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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glutamate site, induced by ATP binding, increases the binding affinity of glutamate. The active site
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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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of GS is located at the subunit interface (which contains n1 & n2) and is constituted mainly by the C domain
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<br>
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of one subunit <font size=1 face ="Arial">(Liaw 1995)</font>.
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<font size=4 face ="Arial">Involving Residues</font><br>
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<B>(insert wiki below that maps the ATP binding)</B></font>
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<br><br>
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<font size=4 face ="Arial">Involving Residues</font>
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<br>
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<font size=2>Most residues involved in enzymatic catalysis are located at the C domain but Asp50 is
<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. The binding of ADP induces Asp50 in order to
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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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enhance the ammonium binding, and then to deprotonate the ammonium ion to form the active ammonia
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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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to attack the gamma-glutamyl phosphate.</font>
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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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<br>
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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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<applet load='1lgr' size='200' color='white' frame='true' align='right' caption='Glutamine Synthetase from Salmonella typhimurium'/>
 
<center><font size=4 face ="Arial">More Catalytic Residues</font><ref name="Liaw"/>
<center><font size=4 face ="Arial">More Catalytic Residues</font><ref name="Liaw"/>
<table border=1>
<table border=1>
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</table>
</table>
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<scene name='User:Grace_Natalie/Involving_residues/1'>Mapping of Involving Residues</scene>
 
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<scene name='User:Grace_Natalie/Gln_synthetase_showing_asp50/1'>Glutamine Synthetase showing ASP50 residue in ATP binding</scene>
 
</center>
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<br><br>

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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