1owc
From Proteopedia
(New page: 200px<br /><applet load="1owc" size="450" color="white" frame="true" align="right" spinBox="true" caption="1owc, resolution 2.2Å" /> '''Three Dimensional Str...) |
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| - | [[Image:1owc.gif|left|200px]]<br /><applet load="1owc" size=" | + | [[Image:1owc.gif|left|200px]]<br /><applet load="1owc" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1owc, resolution 2.2Å" /> | caption="1owc, resolution 2.2Å" /> | ||
'''Three Dimensional Structure Analysis Of The R109L Variant of the Type II Citrate Synthase From E. Coli'''<br /> | '''Three Dimensional Structure Analysis Of The R109L Variant of the Type II Citrate Synthase From E. Coli'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The citrate synthase of Escherichia coli is an example of a Type II | + | The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH. In previous crystallographic work, we defined the NADH binding sites, identifying nine amino acids whose side chains were proposed to make hydrogen bonds with the NADH molecule. Here, we describe the functional properties of nine sequence variants, in which these have been replaced by nonbonding residues. All of the variants show some changes in NADH binding and inhibition and small but significant changes in kinetic parameters for catalysis. In three cases, Y145A, R163L, and K167A, NADH inhibition has become extremely weak. We have used nanospray/time-of-flight mass spectrometry, under non-denaturing conditions, to show that two of these, R163L and K167A, do not form hexamers in response to NADH binding, unlike the wild type enzyme. One variant, R109L, shows tighter NADH binding. We have crystallized this variant and determined its structure, with and without bound NADH. Unexpectedly, the greatest structural changes in the R109L variant are in two regions outside the NADH binding site, both of which, in wild type citrate synthase, have unusually high mobilities as measured by crystallographic thermal factors. In the R109L variant, both regions (residues 260 -311 and 316-342) are much less mobile and have rearranged significantly. We argue that these two regions are elements in the path of communication between the NADH binding sites and the active sites and are centrally involved in the regulatory conformational change in E. coli citrate synthase. |
==About this Structure== | ==About this Structure== | ||
| - | 1OWC is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with SO4 as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Citrate_(Si)-synthase Citrate (Si)-synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.3.1 2.3.3.1] Full crystallographic information is available from [http:// | + | 1OWC is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Citrate_(Si)-synthase Citrate (Si)-synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.3.1 2.3.3.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1OWC OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Brayer, G | + | [[Category: Brayer, G D.]] |
[[Category: Choudhary, K.]] | [[Category: Choudhary, K.]] | ||
| - | [[Category: Donald, L | + | [[Category: Donald, L J.]] |
| - | [[Category: Duckworth, H | + | [[Category: Duckworth, H W.]] |
| - | [[Category: Hultin, P | + | [[Category: Hultin, P G.]] |
[[Category: Maurus, R.]] | [[Category: Maurus, R.]] | ||
| - | [[Category: Nguyen, N | + | [[Category: Nguyen, N T.]] |
[[Category: Sadler, G.]] | [[Category: Sadler, G.]] | ||
| - | [[Category: Stokell, D | + | [[Category: Stokell, D J.]] |
[[Category: SO4]] | [[Category: SO4]] | ||
[[Category: allostery]] | [[Category: allostery]] | ||
| Line 30: | Line 30: | ||
[[Category: type ii citrate synthase]] | [[Category: type ii citrate synthase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:22:19 2008'' |
Revision as of 12:22, 21 February 2008
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Three Dimensional Structure Analysis Of The R109L Variant of the Type II Citrate Synthase From E. Coli
Overview
The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH. In previous crystallographic work, we defined the NADH binding sites, identifying nine amino acids whose side chains were proposed to make hydrogen bonds with the NADH molecule. Here, we describe the functional properties of nine sequence variants, in which these have been replaced by nonbonding residues. All of the variants show some changes in NADH binding and inhibition and small but significant changes in kinetic parameters for catalysis. In three cases, Y145A, R163L, and K167A, NADH inhibition has become extremely weak. We have used nanospray/time-of-flight mass spectrometry, under non-denaturing conditions, to show that two of these, R163L and K167A, do not form hexamers in response to NADH binding, unlike the wild type enzyme. One variant, R109L, shows tighter NADH binding. We have crystallized this variant and determined its structure, with and without bound NADH. Unexpectedly, the greatest structural changes in the R109L variant are in two regions outside the NADH binding site, both of which, in wild type citrate synthase, have unusually high mobilities as measured by crystallographic thermal factors. In the R109L variant, both regions (residues 260 -311 and 316-342) are much less mobile and have rearranged significantly. We argue that these two regions are elements in the path of communication between the NADH binding sites and the active sites and are centrally involved in the regulatory conformational change in E. coli citrate synthase.
About this Structure
1OWC is a Single protein structure of sequence from Escherichia coli with as ligand. Active as Citrate (Si)-synthase, with EC number 2.3.3.1 Full crystallographic information is available from OCA.
Reference
Probing the roles of key residues in the unique regulatory NADH binding site of type II citrate synthase of Escherichia coli., Stokell DJ, Donald LJ, Maurus R, Nguyen NT, Sadler G, Choudhary K, Hultin PG, Brayer GD, Duckworth HW, J Biol Chem. 2003 Sep 12;278(37):35435-43. Epub 2003 Jun 23. PMID:12824188
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