title: ATP hydrolysis and synthesis of a rotary motor V-ATPase from Thermus thermophilus.
abstract: Vacuolar-type H(+)-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site. In contrast, the enzyme was very active for ATP synthesis (approximately 70 s(-1)) with the K(m) values for ADP and phosphate being 4.7 +/- 0.5 and 460 +/- 30 microm, respectively. Single molecule observation showed V-ATPase rotated in a 120 degrees stepwise manner, and analysis of dwelling time allowed the binding rate constant k(on) for ATP to be estimated ( approximately 1.1 x 10(6) m(-1) s(-1)), which was much lower than the k(on) (= V(max)/K(m)) for ADP ( approximately 1.4 x 10(7) m(-1) s(-1)). The slower k(on)(ATP) than k(on)(ADP) and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses.
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nakano
authors.forename: Hiromi
authors.initials: H
authors.lastname: Imamura
authors.forename: Masashi
authors.initials: M
authors.lastname: Toei
authors.forename: Masatada
authors.initials: M
authors.lastname: Tamakoshi
authors.forename: Masasuke
authors.initials: M
authors.lastname: Yoshida
authors.forename: Ken
authors.initials: K
authors.lastname: Yokoyama
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D002384:Catalysis; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008954:Models, Biological; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011522:Protons; D016963:Thermus thermophilus / Q000201:enzymology*; D013997:Time Factors
chemical_list: D008081:Liposomes; D009711:Nucleotides; D010710:Phosphates; D011522:Protons; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium