RAT-LIVER ATP SYNTHASE - RELATIONSHIP OF THE UNIQUE SUBSTRUCTURE OF THE F1 MOIETY TO ITS NUCLEOTIDE-BINDING PROPERTIES, ENZYMATIC STATES, AND CRYSTALLINE FORM

The F-1 moiety of rat liver ATP synthase has a molecular mass of 370,0 00, exhibits the unique substructure alpha(3) beta(3) gamma delta epsi lon, and fully restores ATP synthesis to F-1-depleted membranes, Here we provide new information about rat liver F-1 as it relates to the re lationship of its unique substructure to its nucleotide binding proper ties, enzymatic states, and crystalline form. Seven types of experimen ts were performed in a comprehensive study. First, the capacity of F-1 to bind [H-3]ADP, the substrate for ATP synthesis and [P-32]AMP-PNP ( 5'-adenylyl-beta,gamma-imidodiphosphate), a nonhydrolyzable ATP analog , was quantified, Second, double label experiments were performed to e stablish whether ADP and AMP-PNP bind to the same or different sites. Third, total nucleotide binding was assessed by the luciferin-lucifera se assay. Fourth, F-1 was subfractionated into an alpha gamma and a be ta delta epsilon fraction, both of which were subjected to nucleotide binding assays, Fifth, the nucleotide binding capacity of F-1 was quan tified after undergoing ATP hydrolysis. Sixth, the intensity of the fl uorescence probe pyrene maleimide bound at cu subunits was monitored b efore and after F-1 experienced ATP hydrolysis, Finally, the catalytic activity and nucleotide content of F-1, obtained from crystals being used in x-ray crystallographic studies was determined. The picture of rat liver F-1 that emerges is one of an enzyme molecule that 1) loads nucleotide readily at five sites; 2) requires for catalysis both the a lpha gamma and the beta delta epsilon fractions; 3) directs the revers ible binding of ATP and ADP to different regions of the enzyme's subst ructure; 4) induces inhibition of ATP hydrolysis only after ADP fills at least five sites; and 5) exists in several distinct forms, one an a ctive, symmetrical form, obtained in the presence of ATP and high P-i and on which an x-ray map at 3.6 Angstrom 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 F-1 and also discusse d relative to those reported for bovine heart F-1, which has been crys tallized with inhibitors in an asymmetrical form and has a propensity for binding nucleotides more tightly.