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New page: left|200px<br /><applet load="1lj1" size="450" color="white" frame="true" align="right" spinBox="true" caption="1lj1, resolution 2.00Å" /> '''Crystal structure of... |
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[[Image:1lj1.jpg|left|200px]]<br /><applet load="1lj1" size=" | [[Image:1lj1.jpg|left|200px]]<br /><applet load="1lj1" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1lj1, resolution 2.00Å" /> | caption="1lj1, resolution 2.00Å" /> | ||
'''Crystal structure of Q363F/R402A mutant flavocytochrome c3'''<br /> | '''Crystal structure of Q363F/R402A mutant flavocytochrome c3'''<br /> | ||
==Overview== | ==Overview== | ||
The ability of an arginine residue to function as the active site acid | The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Bronsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Bronsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Bronsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site. | ||
==About this Structure== | ==About this Structure== | ||
1LJ1 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 FUM 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:// | 1LJ1 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=FUM:'>FUM</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=1LJ1 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: Leys, D.]] | [[Category: Leys, D.]] | ||
[[Category: Miles, C | [[Category: Miles, C S.]] | ||
[[Category: Mowat, C | [[Category: Mowat, C G.]] | ||
[[Category: Pankhurst, K | [[Category: Pankhurst, K L.]] | ||
[[Category: Reid, G | [[Category: Reid, G A.]] | ||
[[Category: Walkinshaw, M | [[Category: Walkinshaw, M D.]] | ||
[[Category: FAD]] | [[Category: FAD]] | ||
[[Category: FUM]] | [[Category: FUM]] | ||
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[[Category: fumarate reductase]] | [[Category: fumarate reductase]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:45:22 2008'' | ||
Revision as of 14:45, 21 February 2008
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Crystal structure of Q363F/R402A mutant flavocytochrome c3
OverviewOverview
The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Bronsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Bronsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Bronsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site.
About this StructureAbout this Structure
1LJ1 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.
ReferenceReference
Engineering water to act as an active site acid catalyst in a soluble fumarate reductase., Mowat CG, Pankhurst KL, Miles CS, Leys D, Walkinshaw MD, Reid GA, Chapman SK, Biochemistry. 2002 Oct 8;41(40):11990-6. PMID:12356299
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