NUCLEOTIDE-BINDING SITES ON ESCHERICHIA-COLI F1-ATPASE - SPECIFICITY OF NONCATALYTIC SITES AND INHIBITION AT CATALYTIC SITES BY MGADP

Nucleotide-depleted EcF(1) binds a maximum of two GTP, ATP, or ADP at noncatalytic sites, whereas all three sites can only be filled by a co mbination of nucleoside di- and triphosphates. MgPPi prevents binding of GTP and significantly slows ATP binding, suggesting that noncatalyt ic sites also bind PPi. No binding of GDP at noncatalytic sites could be detected. The slow rate of GTP dissociation from noncatalytic sites (t(1/2) = 175 min) is increased 2-8-fold by EDTA, MgPPi, MgADP, or ED TA/ATP, but 23-fold by conditions for ATP hydrolysis. ATP hydrolysis b y EcF(1), depleted of both its inhibitory epsilon-subunit and endogeno us 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 EcF(1) containing endogenous inhibitory ADP, preincu bation with an ATP-regenerating system results in a burst of activity, whereas the control shows a lag. This same enzyme form shows signific ant inhibition with increasing concentrations of Mg2+ during ATP hydro lysis but lesser levels of inhibition when other NTP substrates are us ed. 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 n ucleotide is bound at catalytic sites as shown by covalent incorporati on of 2-azido-ADP. The results suggest that ligand specificity may not be a reliable means of distinguishing between catalytic and noncataly tic sites and that MgADP inhibition should be taken into account in th e kinetic analysis of EcF(1) mutants.