Kinetic Mechanism of F_o·F₁ Mitochondrial ATPase: Mg²⁺ Requirement for Mg·ATP Hydrolysis

A. V. Syroeshkin, M. A. Galkin, A. V. Sedlov, and A. D. Vinogradov^*

Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (7-095) 939-3955; E-mail: adv@biochem.bio.msu.su

^* To whom correspondence should be addressed.

Received February 4, 1999; Revision received March 9, 1999

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The initial rates of ATP hydrolysis catalyzed by F_o·F₁ (bovine heart submitochondrial particles) preincubated in the presence of P_i 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 on 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_o·F₁-ATPase is discussed.

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KEY WORDS: F_o·F₁-ATP synthase, F₁-ATPase, mitochondria, ATP hydrolysis, oxidative phosphorylation

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