ATP SYNTHESIS BY THE F0F1-ATPASE FROM THE THERMOPHILIC BACILLUS PS3 CO-RECONSTITUTED WITH BACTERIORHODOPSIN INTO LIPOSOMES - EVIDENCE FOR STIMULATION OF ATP SYNTHESIS BY ATP BOUND TO A NONCATALYTIC BINDING-SITE

F-type ATPase from the thermophilic Bacillus PS3, TF0F1, which was essentially free of bound nucleotides after isolation and purification, was co-reconstituted into liposomes with the light-driven proton pump bacteriorhodopsin, The time course of the light-induced ATP synthesis was biphasic; an initial slow phase accelerated to a final steady-state rate two to three times faster, Adding ATP before initiating the reaction suppressed the slow phase, suggesting that the state of occupancy of specific sites by ATP regulated the synthetic activity of TF0F1. Incubating the purified TF0F1 with ADP and ATP revealed one ADP and two ATP binding sites that were stable to gel filtration, We analyzed the time courses of light-induced ATP synthesis for the enzyme with different nucleotide content, after co-reconstitution into liposomes with bacteriorhodopsin. The two ATP sites were identified to have regulatory function. A complex containing TF0F1 . ADP, 1:1, was co-reconstituted with various quantities of ATP to obtain a range of molar ratios of TF0F1 . ADP:ATP of between 1:0 and 1:1.7, It was found that the initial rate of ATP synthesis increased with the level of ATP bound to the enzyme. After binding one ATP, a stimulation of ATP synthesis by a factor of 2 was observed. The second ATP site also exhibited regulatory properties, It stimulated ATP synthesis but to a much smaller extent; the stimulation did not exceed 20%, finding of the photoreactive analogues 2-azido-[alpha-P-32]ADP and 2-azido-[alpha-P-32]ATP to the TF0F1 and their effects on the rate of ATP synthesis are described further, Importantly, after covalent labeling of the enzyme, tryptic digestion, and high performance liquid chromatography purification, the label was found associated with the beta Y364-containing tryptic peptide in all cases, beta Y364 is in the region of conserved residues GXEHYXXA, which is in the beta subunit and known to be part of the noncatalytic site.