Molecular recognition moiety and its target biomolecule interact in switching enzyme activity.
We conjugated a molecular recognition moiety, biotin, with an enzyme site-specifically near to its active site and succeeded in inactivating the enzyme by binding the specific target biomolecule avidin to biotin. Bacterial P450 was used as a model enzyme, which has attracted much attention in several fields. Site-directed mutagenesis was conducted to produce a mutant P450 that could attach biotin site-specifically. The activity of the conjugate decreased markedly to one tenth of that of biotinylated P450 after binding to avidin. Ultraviolet-visible spectroscopy of the carbon monoxide-bound P450, circular dichroism data, and the ratio of the active form to the sum of the active form and the inactive form indicated that this decrease in activity was because of a conformational change in the tertiary structure surrounding the active center after avidin binding, while the secondary structure of P450 remained unchanged.
Chemical Resources Laboratory, Tokyo Institute of Technology, R1-17, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8503, Japan.
Yuhei
Y
Oshiba
Takanori
T
Tamaki
Hidenori
H
Ohashi
Hidehiko
H
Hirakawa
Satoshi
S
Yamaguchi
Teruyuki
T
Nagamune
Takeo
T
Yamaguchi
D001360:Avidin / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D001360:Avidin; D001710:Biotin; D019475:Camphor 5-Monooxygenase