Characterization of the nucleotide tight‐binding sites of the isolated mitochondrial F1‐ATPase

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Abstract
The properties of the nucleotides tightly bound with mitochondrial F1‐ATPase were examined. One of three bound nucleotide molecules is localized at the site with K dR̃10−7 M and released with k off∼0.1 s−1. The second nucleotide molecule is bound with the enzyme with K d ∼10−8 M and k off for its dissociation is 3 × 10−4 s−1. The third is never released even in the presence of 1 mM ATP or ADP. The last two nucleotides are believed to be bound at the noncatalytic sites of F1‐ATPase. Pyrophosphate promotes liberation of two releasable nucleotide molecules, decreasing the affinity of the enzyme to AD(T)P. From the results obtained it follows that the only suitable criterion for localization of the nucleotide at the F1‐ATPase catalytic site is the high rate (k off ≧ 0.1 s−1) of its spontaneous release.