One of the non-exchangeable nucleotides of the mitochondrial F-1-ATPase isbound at a beta-subunit: evidence for a non-rotatory two-site catalytic mechanism

In active MF1, one of the two non-exchangeable tightly bound adenine nucleo tides 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 slow ly dephosphorylated to ADP which is subsequently released, without effect o n activity. When enzyme with about one residual ADP left (at the D-site) wa s incubated at pH 7.3, after dilution of the glycerol, with 400 mu M I [C-1 4]ATP under varying conditions, the amount of tightly bound nucleotide trip hosphate again correlated well with activity, the residual ADP being bound at the D-site. Optimal results were obtained when the incubation was perfor med in the presence of a regenerating system. Binding of 2-azido-ATP instea d of ATP to the T-site as a triphosphate, as indicated by the specific acti vity of the enzyme, appeared to be optimal when the binding was performed a t pH 6.4 in the absence of Mg2+ and with high concentrations of the nucleot ide. Under such conditions, 3 mol 2-azido-AXP per mol Fl remained tightly b ound after ammonium sulfate precipitation and column centrifugation, in add ition to about one residual ADP at the D-sire. After a 2-min period of turn over with ATP/Mg2+ as substrate two mol 2-azido-AXP were left on the enzyme , of which one was bound at a beta-site. These results show that one of the nea-catalytic nucleotide binding sites that contain tightly bound nucleoti des, is a beta-site, in conflict with the requirements for a rotatory tri-s ite mechanism for ATP hydrolysis. This p-site can further be identified wit h the T-site. The validity of these conclusions for F-1 from other sources and for catalysis by membrane-bound enzyme is discussed. (C) 1999 Elsevier Science B.V. All rights reserved.