One of the non-exchangeable nucleotides of the mitochondrial F1-ATPase is bound at a β-subunit: evidence for a non-rotatory two-site catalytic mechanism

AbstractIn active MF1, one of the two non-exchangeable tightly bound adenine nucleotides is an ATP, while the other is an ADP. The respective sites are called the T-site and the D-site. The activity of the enzyme correlates linearly with the amount of bound ATP, ADP at the T-site being inhibitory. When MF1 is stored at room temperature in 50% glycerol and 100 mM Tris-HCl (pH 7.3) after slow passage through a Sephadex column, the tightly bound ATP is slowly dephosphorylated to ADP which is subsequently released, without effect on activity. When enzyme with about one residual ADP left (at the D-site) was incubated at pH 7.3, after dilution of the glycerol, with 400 μM [14C]ATP under varying conditions, the amount of tightly bound nucleotide triphosphate again correlated well with activity, the residual ADP being bound at the D-site. Optimal results were obtained when the incubation was performed in the presence of a regenerating system. Binding of 2-azido-ATP instead of ATP to the T-site as a triphosphate, as indicated by the specific activity of the enzyme, appeared to be optimal when the binding was performed at pH 6.4 in the absence of Mg2+ and with high concentrations of the nucleotide. Under such conditions, 3 mol 2-azido-AXP per mol F1 remained tightly bound after ammonium sulfate precipitation and column centrifugation, in addition to about one residual ADP at the D-site. After a 2-min period of turnover with ATP/Mg2+ as substrate two mol 2-azido-AXP were left on the enzyme, of which one was bound at a β-site. These results show that one of the non-catalytic nucleotide binding sites that contain tightly bound nucleotides, is a β-site, in conflict with the requirements for a rotatory tri-site mechanism for ATP hydrolysis. This β-site can further be identified with the T-site. The validity of these conclusions for F1 from other sources and for catalysis by membrane-bound enzyme is discussed