The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F[sub 1]-ATPase

The recent finding that the presence of ATP at noncatalytic sites of chloroplast F[sub 1]-ATPase (CF[sub 1]) is necessary for ATPase activity prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF[sub 1] containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg[sup 2+] and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF[sub 1] preparations were used for measurement of the time course of nucleotide binding from 10 to 100 [mu]M concentrations of [sup 3]H-labeled ADP, ATP, or GTP. The presence of Mg[sup 2+] strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg[sup 2+] binds ADP with a rate constant of 0.5 [times] 10[sup 6] M[sup [minus]1] min[sup [minus]1] to give an enzyme with two ADP at noncatalytic sites with a K[sub d] of about 0.1 [mu]M. Upon exposure to Mg[sup 2+] and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [[sup 3]H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF[sub 1] is exposed to Mg[sup 2+] and ADP and that can bind ATP rapidly as designated as site A; the site that fills with ATP as ADP dissociates when then enzyme is exposed to Mg[sup 2+] and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF[sub 1] with ADp at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured.