Kinetic mechanism of F0•F1 mitochondrial ATPase:: Mg2+ requirement for Mg•ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by F-0. F-1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg . ATP concentrations, The results suggest the mechanism in 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. Simple hyperbolic Michaelis-Menten dependences of the reaction rate an the substrate (Mg . ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for 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 competes with Mg . ATP, and free Ca2+ does not affect the hydrolysis of Mg . ATP 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 saturation 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 (catalytic turnover) or slow isomerization of the enzyme-product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited 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.