Nucleotide-binding sites on Escherichia coli F1-ATPase. Specificity of noncatalytic sites and inhibition at catalytic sites by MgADP.

Nucleotide-depleted EcF1 binds a maximum of two GTP, ATP, or ADP at noncatalytic sites, whereas all three sites can only be filled by a combination of nucleoside di- and triphosphates. MgPPi prevents binding of GTP and significantly slows ATP binding, suggesting that non-catalytic sites also bind PPi. No binding of GDP at non-catalytic sites could be detected. The slow rate of GTP dissociation from noncatalytic sites (t1/2 = 175 min) is increased 2-8-fold by EDTA, MgPPi, MgADP, or EDTA/ATP, but 23-fold by conditions for ATP hydrolysis. ATP hydrolysis by EcF1, depleted of both its inhibitory epsilon-subunit and endogenous nucleotides, shows a burst of activity. However, it shows a lag if preincubated with MgADP but not when preincubated with Mg2+ alone. For epsilon-depleted EcF1 containing endogenous inhibitory ADP, preincubation with an ATP-regenerating system results in a burst of activity, whereas the control shows a lag. This same enzyme form shows significant inhibition with increasing concentrations of Mg2+ during ATP hydrolysis but lesser levels of inhibition when other NTP substrates are used. With the five-subunit enzyme, increasing amounts of azide cause an increase in the level of inhibition with a corresponding increase in amount of bound nucleotide resistant to rapid chase. Azide-trappable nucleotide is bound at catalytic sites as shown by covalent incorporation of 2-azido-ADP. The results suggest that ligand specificity may not be a reliable means of distinguishing between catalytic and noncatalytic sites and that MgADP inhibition should be taken into account in the kinetic analysis of EcF1 mutants.