1fnj
From Proteopedia
(New page: 200px<br /><applet load="1fnj" size="450" color="white" frame="true" align="right" spinBox="true" caption="1fnj, resolution 1.90Å" /> '''CRYSTAL STRUCTURE AN...) |
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| - | [[Image:1fnj.gif|left|200px]]<br /><applet load="1fnj" size=" | + | [[Image:1fnj.gif|left|200px]]<br /><applet load="1fnj" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1fnj, resolution 1.90Å" /> | caption="1fnj, resolution 1.90Å" /> | ||
'''CRYSTAL STRUCTURE ANALYSIS OF CHORISMATE MUTASE MUTANT C88S/R90K'''<br /> | '''CRYSTAL STRUCTURE ANALYSIS OF CHORISMATE MUTASE MUTANT C88S/R90K'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Combinatorial mutagenesis and in vivo selection experiments previously | + | Combinatorial mutagenesis and in vivo selection experiments previously afforded functional variants of the AroH class Bacillus subtilis chorismate mutase lacking the otherwise highly conserved active site residue Arg(90). Here, we present a detailed kinetic and crystallographic study of several such variants. Removing the arginine side chain (R90G and R90A) reduced catalytic efficiency by more than 5 orders of magnitude. Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand in k(cat). Remarkably, the lysine could be placed at position 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site. Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended conformation that would place its epsilon-ammonium group within hydrogen-bonding distance of the ether oxygen of bound chorismate in the transition state. These results provide support for the hypothesis that developing negative charge in the highly polarized transition state is stabilized electrostatically by a strategically placed cation. The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificial catalysts are discussed. |
==About this Structure== | ==About this Structure== | ||
| - | 1FNJ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis]. Active as [http://en.wikipedia.org/wiki/Chorismate_mutase Chorismate mutase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.4.99.5 5.4.99.5] Full crystallographic information is available from [http:// | + | 1FNJ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis]. Active as [http://en.wikipedia.org/wiki/Chorismate_mutase Chorismate mutase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.4.99.5 5.4.99.5] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1FNJ OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Chorismate mutase]] | [[Category: Chorismate mutase]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Chen, I | + | [[Category: Chen, I A.]] |
[[Category: Grisostomi, C.]] | [[Category: Grisostomi, C.]] | ||
[[Category: Hilvert, D.]] | [[Category: Hilvert, D.]] | ||
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[[Category: x-ray structure]] | [[Category: x-ray structure]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:40:33 2008'' |
Revision as of 10:40, 21 February 2008
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CRYSTAL STRUCTURE ANALYSIS OF CHORISMATE MUTASE MUTANT C88S/R90K
Overview
Combinatorial mutagenesis and in vivo selection experiments previously afforded functional variants of the AroH class Bacillus subtilis chorismate mutase lacking the otherwise highly conserved active site residue Arg(90). Here, we present a detailed kinetic and crystallographic study of several such variants. Removing the arginine side chain (R90G and R90A) reduced catalytic efficiency by more than 5 orders of magnitude. Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand in k(cat). Remarkably, the lysine could be placed at position 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site. Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended conformation that would place its epsilon-ammonium group within hydrogen-bonding distance of the ether oxygen of bound chorismate in the transition state. These results provide support for the hypothesis that developing negative charge in the highly polarized transition state is stabilized electrostatically by a strategically placed cation. The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificial catalysts are discussed.
About this Structure
1FNJ is a Single protein structure of sequence from Bacillus subtilis. Active as Chorismate mutase, with EC number 5.4.99.5 Full crystallographic information is available from OCA.
Reference
A strategically positioned cation is crucial for efficient catalysis by chorismate mutase., Kast P, Grisostomi C, Chen IA, Li S, Krengel U, Xue Y, Hilvert D, J Biol Chem. 2000 Nov 24;275(47):36832-8. PMID:10960481
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