Nucleotide binding of an ADP analog to cooperating sites of chloroplast F1-ATPase (CF1).
Pre-steady state nucleotide binding to the chloroplast F1-ATPase (CF1) was measured in a stopped-flow apparatus by monitoring the change of fluorescence intensity of TNP-ADP upon binding. The analysis of the time courses led to the proposal of a mechanism of nucleotide binding with the following characteristics. (a) It involves three sites binding nucleotides in a concerted manner. (b) Each binding site is able to undergo a conformational change from a loose binding state into a state refraining from any direct release of the bound nucleotide into the medium. Only the reverse reaction via the loose binding state enables release out of the tight binding state. (c) Due to very strong negative cooperativity, a maximum of two of the three sites can be found in the state of tight binding. (d) Cooperativity between the three sites leads to a slower nucleotide binding of the second nucleotide compared to the first nucleotide. Furthermore, the conformational change from the loose binding state to the tight binding state is slowed down if one of the other sites already is in the tight binding state. Three-sites mechanisms in which rotation leads to an exchange of the properties of the binding sites failed to simulate the observed time courses of nucleotide binding. However, as the experimental set up was designed to prevent catalysis taking place, our results entirely agree with the current finding that rotation requires catalytic turnover of the enzyme.
Günther
S
S
ITZ School of Veterinary Medicine, Hannover, Germany.
Huchzermeyer
B
B
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
D009711:Nucleotides; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D006180:Proton-Translocating ATPases