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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 HEM and CAM 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 (Tokyo). 2000 Dec;128(6):965-74. PMID:11098139
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