Origin of apparent negative cooperativity of F1-ATPase

Abstract In order to get insight into the origin of apparent negative cooperativity observed for F 1 -ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F 1 -ATPase, α (W463F)3 β (Y341W)3 γ and α (K175A/T176A/W463F)3 β (Y341W)3 γ. For α (W463F)3 β (Y341W)3 γ, apparent K m 's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent K m 's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 μM). On the other hand, in case of α (K175A/T176A/W463F)3 β (Y341W)3 γ, which lacks noncatalytic nucleotide binding sites, the apparent K m of ATPase activity (10 μM) roughly agreed with the highest K m of fluorescence measurements (27 μM). The results indicate that in case of α (W463F)3 β (Y341W)3 γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K m of ATPase activity does not represent the ATP binding to a catalytic site. In case of α (K175A/T176A/W463F)3 β (Y341W)3 γ, the K m of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie–Hofstee plot of ATPase reaction by α (K175A/T176A/W463F)3 β (Y341W)3 γ was rather linear compared with that of α (W463F)3 β (Y341W)3 γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F 1 -ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K m 's of 100–300 μM and 1–30 μM range for wild-type F 1 -ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.