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

Peter L. Pedersen, Joanne Hullihen, Mario Bianchet, L. Mario Amzel, Michael S. Lebowitz

Research output: Contribution to journalArticlepeer-review

Abstract

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 Pi 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.

Original languageEnglish (US)
Pages (from-to)1775-1784
Number of pages10
JournalJournal of Biological Chemistry
Volume270
Issue number4
DOIs
StatePublished - Jan 27 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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