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Abstract
Systematic studies of the catalytic metal ion dependence and nucleotide dependence of nucleoside-triphosphate hydrolysis catalyzed by beef heart mitochondrial F$\sb1$-ATPase were performed. Divalent metal ions (Ba$\sp{2+}$, Ca$\sp{2+}$, Cd$\sp{2+}$, Co$\sp{2+}$, Cu$\sp{2+}$, Fe$\sp{2+}$, Mg$\sp{2+}$, Mn$\sp{2+}$, Ni$\sp{2+}$, Sr$\sp{2+}$, and Zn$\sp{2+}$) were tested for their effectiveness as cofactors for F$\sb1$ catalysts of the hydrolysis of ATP, ITP, and many other nucleoside-triphosphates. The dependence on the identity of the metal ion of solvent deuterium isotope effects, nitration effects, specific temperature effects, and effects of different inhibitors were studied, as was the nucleotide dependence of the temperature effect on the deuterium solvent isotope effects. Results reveal that temperature-sensitive, metal ion- and nucleotide-dependent parameters regulate F$\sb1$-catalyzed nucleotide hydrolysis, and determine the identity of rate-determining steps and their importance in catalytic regulation. Specific metal ion properties responsible for observed dependencies, include the pK$\sb{\rm a}$ of the metal-coordinated water molecules and the metal-nucleotide stability constants. The results support a catalytic mechanism for F$\sb1$ whereby an active site tyrosinate ion deprotonates a metal-coordinated water molecule in a $\gamma$-monodentate metal-nucleotide complex, leading to bidentate chelation of the metal ion by the nucleotide phosphates and concomitant nucleotide hydrolysis. After formation of the Pi-metal-ADP complex, all metal ion-ADP bonds are broken before product release.
Exchange inert Cr(III) nucleotide complexes and diastereomers of ATP$\alpha$S and ATP$\beta$S were used as inhibitors and substrates respectively of F$\sb1$-catalyzed nucleotide hydrolysis. The results indicated F$\sb1$ is not stereospecific when binding $\beta$,$\gamma$-bidentate metal-nucleotide complexes, and monodentate complexes are better inhibitors of F$\sb1$ than are bidentate complexes, supporting the suggestion that a monodentate metal-ATP complex is the true substrate for F$\sb1$. Results also support the existence of the proposed Pi-metal-ADP intermediate and indicate the catalytic metal ion is chelated to the $\alpha$ and $\beta$ phosphates during the reaction and all bonds between the metal ion and ADP are broken before product release occurs. These bond breaking steps may be rate-determining in some cases.
