1jt9
From Proteopedia
(New page: 200px<br /><applet load="1jt9" size="450" color="white" frame="true" align="right" spinBox="true" caption="1jt9, resolution 2.06Å" /> '''Structure of the mut...) |
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| - | [[Image:1jt9.jpg|left|200px]]<br /><applet load="1jt9" size=" | + | [[Image:1jt9.jpg|left|200px]]<br /><applet load="1jt9" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1jt9, resolution 2.06Å" /> | caption="1jt9, resolution 2.06Å" /> | ||
'''Structure of the mutant F174A T form of the Glucosamine-6-Phosphate deaminase from E.coli'''<br /> | '''Structure of the mutant F174A T form of the Glucosamine-6-Phosphate deaminase from E.coli'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The active site of glucosamine-6-phosphate deaminase from Escherichia coli | + | The active site of glucosamine-6-phosphate deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) has a complex lid formed by two antiparallel beta-strands connected by a helix-loop segment (158-187). This motif contains Arg172, which is a residue involved in binding the substrate in the active-site, and three residues that are part of the allosteric site, Arg158, Lys160 and Thr161. This dual binding role of the motif forming the lid suggests that it plays a key role in the functional coupling between active and allosteric sites. Previous crystallographic work showed that the temperature coefficients of the active-site lid are very large when the enzyme is in its T allosteric state. These coefficients decrease in the R state, thus suggesting that this motif changes its conformational flexibility as a consequence of the allosteric transition. In order to explore the possible connection between the conformational flexibility of the lid and the function of the deaminase, we constructed the site-directed mutant Phe174-Ala. Phe174 is located at the C-end of the lid helix and its side-chain establishes hydrophobic interactions with the remainder of the enzyme. The crystallographic structure of the T state of Phe174-Ala deaminase, determined at 2.02 A resolution, shows no density for the segment 162-181, which is part of the active-site lid (PDB 1JT9). This mutant form of the enzyme is essentially inactive in the absence of the allosteric activator, N-acetylglucosamine-6-P although it recovers its activity up to the wild-type level in the presence of this ligand. Spectrometric and binding studies show that inactivity is due to the inability of the active-site to bind ligands when the allosteric site is empty. These data indicate that the conformational flexibility of the active-site lid critically alters the binding properties of the active site, and that the occupation of the allosteric site restores the lid conformational flexibility to a functional state. |
==About this Structure== | ==About this Structure== | ||
| - | 1JT9 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Active as [http://en.wikipedia.org/wiki/Glucosamine-6-phosphate_deaminase Glucosamine-6-phosphate deaminase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.99.6 3.5.99.6] Full crystallographic information is available from [http:// | + | 1JT9 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Active as [http://en.wikipedia.org/wiki/Glucosamine-6-phosphate_deaminase Glucosamine-6-phosphate deaminase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.99.6 3.5.99.6] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1JT9 OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Bustos-Jaimes, I.]] | [[Category: Bustos-Jaimes, I.]] | ||
| - | [[Category: Calcagno, M | + | [[Category: Calcagno, M L.]] |
[[Category: Horjales, E.]] | [[Category: Horjales, E.]] | ||
[[Category: Rudino-Pinera, E.]] | [[Category: Rudino-Pinera, E.]] | ||
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[[Category: structural flexibility]] | [[Category: structural flexibility]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:26:27 2008'' |
Revision as of 11:26, 21 February 2008
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Structure of the mutant F174A T form of the Glucosamine-6-Phosphate deaminase from E.coli
Overview
The active site of glucosamine-6-phosphate deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) has a complex lid formed by two antiparallel beta-strands connected by a helix-loop segment (158-187). This motif contains Arg172, which is a residue involved in binding the substrate in the active-site, and three residues that are part of the allosteric site, Arg158, Lys160 and Thr161. This dual binding role of the motif forming the lid suggests that it plays a key role in the functional coupling between active and allosteric sites. Previous crystallographic work showed that the temperature coefficients of the active-site lid are very large when the enzyme is in its T allosteric state. These coefficients decrease in the R state, thus suggesting that this motif changes its conformational flexibility as a consequence of the allosteric transition. In order to explore the possible connection between the conformational flexibility of the lid and the function of the deaminase, we constructed the site-directed mutant Phe174-Ala. Phe174 is located at the C-end of the lid helix and its side-chain establishes hydrophobic interactions with the remainder of the enzyme. The crystallographic structure of the T state of Phe174-Ala deaminase, determined at 2.02 A resolution, shows no density for the segment 162-181, which is part of the active-site lid (PDB 1JT9). This mutant form of the enzyme is essentially inactive in the absence of the allosteric activator, N-acetylglucosamine-6-P although it recovers its activity up to the wild-type level in the presence of this ligand. Spectrometric and binding studies show that inactivity is due to the inability of the active-site to bind ligands when the allosteric site is empty. These data indicate that the conformational flexibility of the active-site lid critically alters the binding properties of the active site, and that the occupation of the allosteric site restores the lid conformational flexibility to a functional state.
About this Structure
1JT9 is a Single protein structure of sequence from Escherichia coli. Active as Glucosamine-6-phosphate deaminase, with EC number 3.5.99.6 Full crystallographic information is available from OCA.
Reference
On the role of the conformational flexibility of the active-site lid on the allosteric kinetics of glucosamine-6-phosphate deaminase., Bustos-Jaimes I, Sosa-Peinado A, Rudino-Pinera E, Horjales E, Calcagno ML, J Mol Biol. 2002 May 24;319(1):183-9. PMID:12051945
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