title: Catalytic site occupancy during ATP hydrolysis by MF1-ATPase. Evidence for alternating high affinity sites during steady-state turnover.
abstract: The mechanism of ATP hydrolysis by the solubilized mitochondrial ATPase (MF1) has been studied under conditions where catalytic turnover occurs at one site, uni-site catalysis (obtained when enzyme is in excess of substrate), or at two sites, bi-site catalysis (obtained when substrate is in excess of enzyme). Pulse-chase experiments support the conclusion that the sites which participate in bi-site catalysis are the same as those which participate in uni-site catalysis. Upon addition of ATP in molar excess to MF1, label that was bound under uni-site conditions dissociates at a rate equal to the rate of bi-site catalysis. Similarly, when medium ATP is removed, label that was bound under bi-site conditions dissociates at a rate equal to the rate of uni-site catalysis. Evidence that a high affinity catalytic site equivalent to the one observed under uni-site conditions participates as an intermediate in bi-site catalysis includes the demonstration of full occupancy of a catalytically competent site during steady-state turnover at nanomolar concentrations of ATP. Improved measurements of the interaction of ADP at a high affinity catalytic site have lead to the revision of several of the rate constants that define uni-site catalysis. The rate constant for unpromoted dissociation of ADP is equal to that for Pi (4 X 10(-3) s-1). The rate of binding ADP at a high affinity chaseable site (Kd = 1 nM) is equal to the rate of binding ATP (4 X 10(6) M-1 s-1). The rate of catalysis obtained when substrate binding at one site promotes product release from an adjacent site (bi-site catalysis) is up to 100,000-fold faster than unpromoted product release (uni-site catalysis).
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse 13210.
authors.forename: D
authors.initials: D
authors.lastname: Cunningham
authors.forename: R L
authors.initials: RL
authors.lastname: Cross
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007700:Kinetics; D008433:Mathematics; D008929:Mitochondria, Heart / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases