Origin of apparent negative cooperativity of F1-ATPase |
| |
Authors: | Sakurako Ono Jun Hirao Hiroyuki Noji Eiro Muneyuki |
| |
Affiliation: | a Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan b Department of Biochemistry, Kanazawa Medical University, Uchinada-cho, Ishikawa 920-02, Japan c Institute of Industrial Science, University of Tokyo, Tokyo 153-8505, Japan |
| |
Abstract: | In order to get insight into the origin of apparent negative cooperativity observed for F1-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F1-ATPase, α(W463F)3β(Y341W)3γ and α(K175A/T176A/W463F)3β(Y341W)3γ. For α(W463F)3β(Y341W)3γ, apparent Km's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent Km'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)3β(Y341W)3γ, which lacks noncatalytic nucleotide binding sites, the apparent Km of ATPase activity (10 μM) roughly agreed with the highest Km of fluorescence measurements (27 μM). The results indicate that in case of α(W463F)3β(Y341W)3γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent Km of ATPase activity does not represent the ATP binding to a catalytic site. In case of α(K175A/T176A/W463F)3β(Y341W)3γ, the Km 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)3β(Y341W)3γ was rather linear compared with that of α(W463F)3β(Y341W)3γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F1-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed Km's of 100-300 μM and 1-30 μM range for wild-type F1-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively. |
| |
Keywords: | F1-ATPase ATP binding Catalysis Noncatalytic site Tryptophan fluorescence |
本文献已被 ScienceDirect 等数据库收录! |
|