1q9i
From Proteopedia
(New page: 200px<br /><applet load="1q9i" size="450" color="white" frame="true" align="right" spinBox="true" caption="1q9i, resolution 1.60Å" /> '''The A251C:S430C doub...) |
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| - | [[Image:1q9i.jpg|left|200px]]<br /><applet load="1q9i" size=" | + | [[Image:1q9i.jpg|left|200px]]<br /><applet load="1q9i" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1q9i, resolution 1.60Å" /> | caption="1q9i, resolution 1.60Å" /> | ||
'''The A251C:S430C double mutant of flavocytochrome c3 from Shewanella frigidimarina'''<br /> | '''The A251C:S430C double mutant of flavocytochrome c3 from Shewanella frigidimarina'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The crystal structures of various different members of the family of | + | The crystal structures of various different members of the family of fumarate reductases and succinate dehydrogenases have allowed the identification of a mobile clamp (or capping) domain [e.g., Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], which has been proposed to be involved in regulating accessibility of the active site to substrate. To investigate this, we have constructed the A251C:S430C double mutant form of the soluble flavocytochrome c(3) fumarate reductase from Shewanella frigidimarina, to introduce an interdomain disulfide bond between the FAD-binding and clamp domains of the enzyme, thus restricting relative mobility between the two. Here, we describe the kinetic and crystallographic analysis of this double mutant enzyme. The 1.6 A resolution crystal structure of the A251C:S430C enzyme under oxidizing conditions reveals the formation of a disulfide bond, while Ellman analysis confirms its presence in the enzyme in solution. Kinetic analyses with the enzyme in both the nonbridged (free thiol) and the disulfide-bridged states indicate a slight decrease in the rate of fumarate reduction when the disulfide bridge is present, while solvent-kinetic-isotope studies indicate that in both wild-type and mutant enzymes the reaction is rate limited by proton and/or hydride transfer during catalysis. The limited effects of the inhibition of clamp domain mobility upon the catalytic reaction would indicate that such mobility is not essential for the regulation of substrate access or product release. |
==About this Structure== | ==About this Structure== | ||
| - | 1Q9I is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Shewanella_frigidimarina Shewanella frigidimarina] with NA, HEM, FAD and TEO as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Succinate_dehydrogenase Succinate dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.3.99.1 1.3.99.1] Full crystallographic information is available from [http:// | + | 1Q9I is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Shewanella_frigidimarina Shewanella frigidimarina] with <scene name='pdbligand=NA:'>NA</scene>, <scene name='pdbligand=HEM:'>HEM</scene>, <scene name='pdbligand=FAD:'>FAD</scene> and <scene name='pdbligand=TEO:'>TEO</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Succinate_dehydrogenase Succinate dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.3.99.1 1.3.99.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1Q9I OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Succinate dehydrogenase]] | [[Category: Succinate dehydrogenase]] | ||
| - | [[Category: Chapman, S | + | [[Category: Chapman, S K.]] |
| - | [[Category: Miles, C | + | [[Category: Miles, C S.]] |
| - | [[Category: Mowat, C | + | [[Category: Mowat, C G.]] |
| - | [[Category: Reid, G | + | [[Category: Reid, G A.]] |
| - | [[Category: Rothery, E | + | [[Category: Rothery, E L.]] |
| - | [[Category: Walkinshaw, M | + | [[Category: Walkinshaw, M D.]] |
[[Category: FAD]] | [[Category: FAD]] | ||
[[Category: HEM]] | [[Category: HEM]] | ||
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[[Category: flavocytochrome; fumarate reductase; disulfide]] | [[Category: flavocytochrome; fumarate reductase; disulfide]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:37:24 2008'' |
Revision as of 12:37, 21 February 2008
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The A251C:S430C double mutant of flavocytochrome c3 from Shewanella frigidimarina
Overview
The crystal structures of various different members of the family of fumarate reductases and succinate dehydrogenases have allowed the identification of a mobile clamp (or capping) domain [e.g., Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], which has been proposed to be involved in regulating accessibility of the active site to substrate. To investigate this, we have constructed the A251C:S430C double mutant form of the soluble flavocytochrome c(3) fumarate reductase from Shewanella frigidimarina, to introduce an interdomain disulfide bond between the FAD-binding and clamp domains of the enzyme, thus restricting relative mobility between the two. Here, we describe the kinetic and crystallographic analysis of this double mutant enzyme. The 1.6 A resolution crystal structure of the A251C:S430C enzyme under oxidizing conditions reveals the formation of a disulfide bond, while Ellman analysis confirms its presence in the enzyme in solution. Kinetic analyses with the enzyme in both the nonbridged (free thiol) and the disulfide-bridged states indicate a slight decrease in the rate of fumarate reduction when the disulfide bridge is present, while solvent-kinetic-isotope studies indicate that in both wild-type and mutant enzymes the reaction is rate limited by proton and/or hydride transfer during catalysis. The limited effects of the inhibition of clamp domain mobility upon the catalytic reaction would indicate that such mobility is not essential for the regulation of substrate access or product release.
About this Structure
1Q9I is a Single protein structure of sequence from Shewanella frigidimarina with , , and as ligands. Active as Succinate dehydrogenase, with EC number 1.3.99.1 Full crystallographic information is available from OCA.
Reference
Probing domain mobility in a flavocytochrome., Rothery EL, Mowat CG, Miles CS, Mott S, Walkinshaw MD, Reid GA, Chapman SK, Biochemistry. 2004 May 4;43(17):4983-9. PMID:15109257
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