INTERACTION OF NUCLEOTIDES WITH CHLOROPLAST COUPLING FACTOR-1

The interactions of ADP, adenylyl imidodiphosphate (AMP-PNP), and ATP with solubilized chloroplast coupling factor (CF1), a latent ATPase, have been studied with equilibrium and kinetic methods. After extensive Sephadex chromatography, the purified CF1 contains 1 mol of ADP/mol of CF1 no other tightly bound nucleotides are found. Radioactively labeled ADP and AMP-PNP can be exchanged for the tightly bound ADP on CF1 and on 7-chloro-4-nitrobenz-2-oxa-l, 3-diazole (NBD) modified CF1 with a characteristic half-time of ~4 h at 25 °C, pH 8.0. This ADP exchange reaction requires binding of ADP at one or more sites on CF1 and proceeds ~4 times faster when heat-activated or NBD-modified heat-activated CF1 is used. The exchange of AMP-PNP for tightly bound ADP requires Mg2+ or Ca2+. At 25 °C the incorporation of 1 mol of [γ-32P,3H]ATP/mol of latent enzyme and hydrolysis into tightly bound [3H]ADP and 32P1 occur within 10 min in the presence of 5 mM Mg2+; ADP strongly inhibits ATP incorporation. The dissociation of Pi is slow (half-time ~24 h), and replacement of the tightly bound ADP by ATP is prevented by bound Pi. In the presence of 2 mM EDTA, incorporation of ATP is very slow and requires the binding of ATP at one or more sites on CF,. Binding studies with forced dialysis indicate the presence of one tight binding site per CF1 for ATP for both the latent and NBD-modified latent enzyme in the presence of 5 mM Mg2+. With 2 mM EDTA, approximately three relatively weak binding sites are found. Incorporation of 1 mol of [7-32P,3H]ATP/mol of heat-activated CF, occurs at 37 °C within 30 s in the presence of 5 mM Ca2+, where the ATPase activity is large; the P1 is completely dissociated, and net catalysis of ATP hydrolysis occurs. Under conditions where the enzyme has a very weak ATPase activity, the P1 is considerably less dissociated. These results suggest a minimum of three nucleotide binding sites on the enzyme. Possible mechanisms for ATP hydrolysis include a single catalytic site with two regulatory nucleotide sites, alternating catalytic sites, and various combinations of these two mechanisms involving three or more nucleotide binding sites. © 1979, American Chemical Society. All rights reserved.