title: Bi-site catalysis in F1-ATPase: does it exist?
abstract: The mechanism of action of F(1)F(0)-ATP synthase is controversial. Some favor a tri-site mechanism, where substrate must fill all three catalytic sites for activity, others a bi-site mechanism, where one of the three sites is always unoccupied. New approaches were applied to examine this question. First, ITP was used as hydrolysis substrate; lower binding affinities of ITP versus ATP enable more accurate assessment of sites occupancy. Second, distributions of all eight possible enzyme species (with zero, one, two or three sites filled) as fraction of total enzyme population at each ITP concentration were calculated, and compared with measured ITPase activity. Confirming data were obtained with ATP as substrate. Third, we performed a theoretical analysis of possible bi-site mechanisms. The results argue convincingly that bi-site hydrolysis activity is negligible, and may not even exist. Effectively, tri-site hydrolysis is the only mechanism. We argue that only tri-site hydrolysis drives subunit rotation. Theoretical analyses of possible bi-site mechanisms reveal serious flaws, not previously recognized. One is that, in bi-site catalysis, the predicted direction of subunit rotation is the same for both ATP synthesis and hydrolysis; a second is that infrequently occurring enzyme species are required.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: J
authors.initials: J
authors.lastname: Weber
authors.forename: A E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D007293:Inosine Triphosphate / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism; D000097630:Inosine Triphosphatase
chemical_list: D000255:Adenosine Triphosphate; D007293:Inosine Triphosphate; D006180:Proton-Translocating ATPases; D011755:Pyrophosphatases; D000097630:Inosine Triphosphatase