X-Ray Crystal Structure and Catalytic Properties of Thr252Ile Mutant of Cytochrome P450cam: Roles of Thr252 and Water in the Active Center
| Autor: | Shingo Nagano, Sam-Yong Park, Yasukazu Kanamori, Yoshitsugu Shiro, Takako Hishiki, Shin-ichi Adachi, Hideo Shimada, Yuzuru Ishimura, Tsuyoshi Egawa, Ryu Makino |
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| Rok vydání: | 2000 |
| Předmět: |
Models
Molecular Threonine Camphor 5-Monooxygenase Cytochrome Protein Conformation Stereochemistry Crystallography X-Ray Biochemistry Catalysis Electron Transport Active center medicine Isoleucine Molecular Biology Ternary complex chemistry.chemical_classification Binding Sites biology Chemistry Water Active site General Medicine Monooxygenase Electron transport chain Camphor Oxygen Crystallography Enzyme Mutagenesis Site-Directed biology.protein Ferric Spectrophotometry Ultraviolet medicine.drug |
| Zdroj: | Journal of Biochemistry. 128:965-974 |
| ISSN: | 0021-924X |
| DOI: | 10.1093/oxfordjournals.jbchem.a022848 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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