Rat liver ATP synthase: Relationship of the unique substructure of the F₁ moiety to its nucleotide binding properties, enzymatic states, and crystalline form

The F₁ moiety of rat liver ATP synthase has a molecular mass of 370,000, exhibits the unique substructure α₃β₃γβ∈, and fully restores ATP synthesis to F₁-depleted membranes. Here we provide new information about rat liver F₁ as it relates to the relationship of its unique substructure to its nucleotide binding properties, enzymatic states, and crystalline form. Seven types of experiments were performed in a comprehensive study. First, the capacity of F₁ to bind [³H]ADP, the substrate for ATP synthesis and [³²P]AMP-PNP (5′-adenylyl-β,γ-imidodiphosphate), a nonhydrolyzable ATP analog, was quantified. Second, double-label experiments were performed to establish whether ADP and AMP-PNP bind to the same or different sites. Third, total nucleotide binding was assessed by the luciferin-luciferase assay. Fourth, F₁ was subfractionated into an αγ and a βδ∈ fraction, both of which were subjected to nucleotide binding assays. Fifth, the nucleotide binding capacity of F₁ was quantified after undergoing ATP hydrolysis. Sixth, the intensity of the fluorescence probe pyrene maleimide bound at α subunits was monitored before and after F₁ experienced ATP hydrolysis. Finally, the catalytic activity and nucleotide content of F₁ obtained from crystals being used in x-ray crystallographic studies was determined. The picture of rat liver F₁ that emerges is one of an enzyme molecule that 1) loads nucleotide readily at five sites; 2) requires for catalysis both the αγ and the βδ∈ fractions; 3) directs the reversible binding of ATP and ADP to different regions of the enzyme's substructure; 4) induces inhibition of ATP hydrolysis only after ADP fills at least five sites; and 5) exists in several distinct forms, one an active, symmetrical form, obtained in the presence of ATP and high P_i and on which an x-ray map at 3.6 Å has been reported (Bianchet, M., Ysern, X., Hullihen, J., Pedersen, P. L., and Amzel, L. M. (1991) J.Biol. Chem. 266, 21197-21201). These results are discussed within the context of a multistate model for rat liver F₁ and also discussed relative to those reported for bovine heart F₁, which has been crystallized with inhibitors in an asymmetrical form and has a propensity for binding nucleotides more tightly.