1geb: Difference between revisions

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X-RAY CRYSTAL STRUCTURE AND CATALYTIC PROPERTIES OF THR252ILE MUTANT OF CYTOCHROME P450CAM

OverviewOverview

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 StructureAbout 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.

ReferenceReference

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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-[[Image:1geb.gif|left|200px]]<br /><applet load="1geb" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="1geb, resolution 2.03&Aring;" />
 '''X-RAY CRYSTAL STRUCTURE AND CATALYTIC PROPERTIES OF THR252ILE MUTANT OF CYTOCHROME P450CAM'''<br />
 ==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.
+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==
-GEB 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://ispc.weizmann.ac.il/oca-bin/ocashort?id=1GEB OCA].
+GEB 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==
-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 (Tokyo). 2000 Dec;128(6):965-74. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=11098139 11098139]
+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: Pseudomonas putida]]
@@ Line 17: / Line 17: @@
 [[Category: Ishimura, Y.]]
 [[Category: Nagano, S.]]
-[[Category: Park, S.Y.]]
+[[Category: Park, S Y.]]
 [[Category: Shimada, H.]]
 [[Category: CAM]]
@@ Line 24: / Line 24: @@
 [[Category: monooxygenase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 15:58:53 2007''
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