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J. Biol. Chem., Vol. 261, Issue 27, 12544-12549, 09, 1986

Adenine nucleotide-binding sites on beef heart F1-ATPase. Conditions that affect occupancy of catalytic and noncatalytic sites

FA Kironde and RL Cross

Beef heart mitochondrial F1 contains a total of six adenine nucleotide- binding sites including at least two different types of sites. Three "exchangeable" sites exchange rapidly during hydrolysis of MgATP, whereas three "nonexchangeable" sites do not (Cross, R. L. and Nalin, C. M. (1982) J. Biol. Chem. 257, 2874-2881). When F1 that has been stored as a suspension in (NH4)2SO4/ATP/EDTA/sucrose/Tris, pH 8.0, is pelleted, rinsed with (NH4)2SO4, dissolved, and desalted, it retains three bound adenine nucleotides. We find that two of these endogenous nucleotides are bound at nonexchangeable sites and one at an exchangeable site. The vacant nonexchangeable site is highly specific for adenine nucleotide and is rapidly filled by ADP upon addition of ADP or during ATP hydrolysis. ADP bound at this site can be removed by reprecipitating the enzyme with (NH4)2SO4. The single nucleotide retained by desalted F1 at an exchangeable site is displaced during hydrolysis of ATP, GTP, or ITP. The binding of PPi at two sites on the enzyme also promotes its dissociation. Neither procedure affects retention of nucleotide at the nonexchangeable sites. These observations, combined with the finding that PPi is much more easily removed from exchangeable sites than ADP, have led to the development of a procedure for preparing F1 with uniform and well-defined nucleotide site occupancy. This involves sequential exposure to MgATP, PPi, and high concentrations of Pi. Unbound ligand is removed between each step. The resulting enzyme, F1[3,0], has three occupied nonexchangeable sites and three vacant exchangeable sites. Evidence that nonexchangeable and exchangeable sites represent noncatalytic and catalytic sites, respectively, suggest that this form of the enzyme will prove useful in numerous applications, including transient kinetic measurements and affinity labeling of active site residues.
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