The mechanism of stimulation of MgATPase activity of chloroplast F 1 ‐ATPase by non‐catalytic adeninenucleotide binding

The presence of ATP at non‐catalytic sites of the chloroplast F 1 ‐ATPase (CF 1 ) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266 , 11551–11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF 1 . Results reported here show that when assayed in the presence of sulfite, CF 1 , with some non‐catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non‐catalytic sites is not necessary for catalytic turnover of CF 1 . However, even though CF 1 with empty non‐catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non‐catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF 1 results from the presence of sulfite, with or without nucleotide binding at non‐catalytic sites, the K m remains constant, at about 50 γM for MgATP and 400 γM for MgGTP. The results obtained show that the ATPase activity of CF 1 is determined by the fraction of the active enzyme. The inactive CF 1 · ADP · Mg 2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF 1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non‐catalytic sites promotes the ATP‐dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non‐catalytic sites results from increasing the portion of CF 1 in an active state during steady‐state ATP hydrolysis.