title: Probing substrate binding site of the Escherichia coli quinol oxidases using synthetic ubiquinol analogues.
abstract: Substrate binding sites of the Escherichia coli bo- and bd-type quinol oxidases were probed with systematically synthesized ubiquinol analogues. The apparent Km values of ubiquinol-2 derivatives to the bo-type enzyme were much lower than that of the corresponding 6-n-decyl derivatives. The isoprenoid structure is less hydrophobic than the saturated n-alkyl group with the same carbon number; therefore, the native isoprenoid side chain appears to play a specific role in quinol binding besides simply increasing hydrophobicity of the molecule. The Vmax values of 2-methoxy-3-ethoxy analogues were greater than that of 2-ethoxy-3-methoxy analogues irrespective of the side chain structure. This result indicates not only that a methoxy group in the 2-position is recognized more strictly than the 3-position by the binding site but also that the side chain structure does not affect binding of the quinol ring moiety. Systematic analysis of the electron-donating activities of the analogues with different substituents in the 5-position revealed that the 5-methyl group is important for the activity. In the parallel studies with the bd-type enzyme, we obtained similar observations except that almost all quinol analogues, but not ubiquinol-1, elicited a remarkable substrate inhibition at higher concentrations. These results indicate that the two structurally unrelated terminal oxidases share common structural properties for the quinol-oxidation site.
authors.affiliation: Department of Agricultural Chemistry, Kyoto University, Sakyo-ku, Kyoto 606, Japan. miyoshi@kais.kyoto-u.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Sakamoto
authors.forename: H
authors.initials: H
authors.lastname: Miyoshi
authors.forename: K
authors.initials: K
authors.lastname: Takegami
authors.forename: T
authors.initials: T
authors.lastname: Mogi
authors.forename: Y
authors.initials: Y
authors.lastname: Anraku
authors.forename: H
authors.initials: H
authors.lastname: Iwamura
mesh_terms: D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D014451:Ubiquinone; D010088:Oxidoreductases; C020094:duroquinol oxidase; C003741:ubiquinol