Design and synthesis of photochemically controllable restriction endonuclease BamHI by manipulating the salt-bridge network in the dimer interface.
The strategy for the design of photochemically controllable enzymes by manipulating the dimer interface is described. Employing a restriction endonuclease BamHI, the selective incorporation of amino acids having a photoremovable 6-nitroveratryl group into the specific position (Lys132) in the dimer interface of the BamHI mutant (H133A) was performed. The activity of the photofunctionalized BamHI mutant was significantly suppressed, and the following photoirradiation induced the recovery of the activity. In addition, uncaging of the 6-nitroveratryl group introduced to Lys132 did not seriously reduce the catalytic activity and affinity for the substrate. These results indicate that the activity of the enzyme can be effectively regulated by caging and uncaging of the specific amino acid in the dimer interface using the photoremovable group.
Endo
Masayuki
M
Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
Nakayama
Koji
K
Majima
Tetsuro
T
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D004247:DNA; D015245:Deoxyribonuclease BamHI / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D011487:Protein Conformation; D012343:RNA, Transfer; D013379:Substrate Specificity
D000596:Amino Acids; D004247:DNA; D012343:RNA, Transfer; D015245:Deoxyribonuclease BamHI