1geb
From Proteopedia
(New page: 200px<br /><applet load="1geb" size="450" color="white" frame="true" align="right" spinBox="true" caption="1geb, resolution 2.03Å" /> '''X-RAY CRYSTAL STRUCT...) |
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| - | [[Image:1geb.gif|left|200px]]<br /><applet load="1geb" size=" | + | [[Image:1geb.gif|left|200px]]<br /><applet load="1geb" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1geb, resolution 2.03Å" /> | caption="1geb, resolution 2.03Å" /> | ||
'''X-RAY CRYSTAL STRUCTURE AND CATALYTIC PROPERTIES OF THR252ILE MUTANT OF CYTOCHROME P450CAM'''<br /> | '''X-RAY CRYSTAL STRUCTURE AND CATALYTIC PROPERTIES OF THR252ILE MUTANT OF CYTOCHROME P450CAM'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The structure-function relationship in cytochrome P450cam monooxygenase | + | The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile. X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H(2)O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators. |
==About this Structure== | ==About this Structure== | ||
| - | 1GEB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_putida Pseudomonas putida] with HEM and CAM as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Camphor_5-monooxygenase Camphor 5-monooxygenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.15.1 1.14.15.1] Full crystallographic information is available from [http:// | + | 1GEB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_putida Pseudomonas putida] with <scene name='pdbligand=HEM:'>HEM</scene> and <scene name='pdbligand=CAM:'>CAM</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Camphor_5-monooxygenase Camphor 5-monooxygenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.15.1 1.14.15.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1GEB OCA]. |
==Reference== | ==Reference== | ||
| - | X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center., Hishiki T, Shimada H, Nagano S, Egawa T, Kanamori Y, Makino R, Park SY, Adachi S, Shiro Y, Ishimura Y, J Biochem | + | X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center., Hishiki T, Shimada H, Nagano S, Egawa T, Kanamori Y, Makino R, Park SY, Adachi S, Shiro Y, Ishimura Y, J Biochem. 2000 Dec;128(6):965-74. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=11098139 11098139] |
[[Category: Camphor 5-monooxygenase]] | [[Category: Camphor 5-monooxygenase]] | ||
[[Category: Pseudomonas putida]] | [[Category: Pseudomonas putida]] | ||
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[[Category: Ishimura, Y.]] | [[Category: Ishimura, Y.]] | ||
[[Category: Nagano, S.]] | [[Category: Nagano, S.]] | ||
| - | [[Category: Park, S | + | [[Category: Park, S Y.]] |
[[Category: Shimada, H.]] | [[Category: Shimada, H.]] | ||
[[Category: CAM]] | [[Category: CAM]] | ||
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[[Category: monooxygenase]] | [[Category: monooxygenase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:49:09 2008'' |
Revision as of 10:49, 21 February 2008
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X-RAY CRYSTAL STRUCTURE AND CATALYTIC PROPERTIES OF THR252ILE MUTANT OF CYTOCHROME P450CAM
Overview
The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile. X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H(2)O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.
About this Structure
1GEB is a Single protein structure of sequence from Pseudomonas putida with and as ligands. Active as Camphor 5-monooxygenase, with EC number 1.14.15.1 Full crystallographic information is available from OCA.
Reference
X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center., Hishiki T, Shimada H, Nagano S, Egawa T, Kanamori Y, Makino R, Park SY, Adachi S, Shiro Y, Ishimura Y, J Biochem. 2000 Dec;128(6):965-74. PMID:11098139
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