X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center.
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.
Hishiki
T
T
Department of Biochemistry, School of Medicine, Keio University, Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan.
Shimada
H
H
Nagano
S
S
Egawa
T
T
Kanamori
Y
Y
Makino
R
R
Park
S Y
SY
Adachi
S
S
Shiro
Y
Y
Ishimura
Y
Y
D001665:Binding Sites; D002164:Camphor / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D004579:Electron Transport; D007532:Isoleucine / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013912:Threonine / Q000378:metabolism*; D014867:Water / Q000378:metabolism*
D007532:Isoleucine; D014867:Water; D013912:Threonine; D002164:Camphor; D019475:Camphor 5-Monooxygenase; D010100:Oxygen