Kinetic mechanism of F-0 center dot F-1 mitochondrial ATPase: Mg2+ requirement for Mg center dot ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by F-0. F-1 (bovine heart sub mitochondrial particles) preincubated in the presence of Pi for complete ac tivation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg . ATP concentrations, The results suggest the mechanism i n which Mg . ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Si mple hyperbolic Michaelis-Menten dependences of the reaction rate an the su bstrate (Mg . ATP) and activating Mg2+ were found. In contrast to the gener ally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibit ion of the reaction by free ATP is due to a decrease of free Mg2+ needed fo r the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca . ATP complex is neither hydrolyzed nor comp etes with Mg . ATP, and free Ca2+ does not affect the hydrolysis of Mg . AT P complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent K-m for Mg . ATP on sa turation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely P-i) thus promoting either release of ADP (cat alytic turnover) or slow isomerization of the enzyme-product complex (forma tion of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg ATP hydrol ysis only slightly depends on pH at saturating Mg2+. In the presence of lim ited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pK(a) 7,5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in F-0. F-1-ATPase is discussed.