Differentiation of the nucleotide-binding sites on nucleotide-depleted mitochondrial F1-ATPase by means of a fluorescent ADP analogue.
The interaction of the fluorescent ADP analogue lin-benzo-ADP (containing a linearly extended version of adenine, in which a benzene ring is inserted between pyrimidine and imidazole ring) with nucleotide-depleted mitochondrial F1 was investigated. It was found that lin-benzo-ADP is able to occupy all six nucleotide-binding sites present on the enzyme. Two sites exhibit a very high affinity for the analogue (dissociation constant, Kd, less than 10 nM) and bind it rapidly (association rate constant, k+1, about 1.10(6) M-1 S-1). A third site shows a lower affinity for the analogue (Kd = 1-2 microM) and is occupied relatively fast (k+1 approximately 10(4) M-1 S-1). Binding of lin-benzo-ADP to these three sites is prevented not only in the presence of excess ADP and ATP, but also by IDP and ITP, thus indicating that these sites are the catalytic ones. As it will be discussed, this conclusion is further corroborated by the finding that release of the analogue from the two high affinity sites can be promoted by binding of nucleoside di- and triphosphates to the third site. The remaining three sites were found to bind lin-benzo-ADP with identical affinity (Kd = 1-2 microM) and with a rather low association rate (k+1 = 300-600 M-1 S-1). Binding of the analogue to them is only prevented by ADP and ATP, but not by IDP and ITP, which confirms that these sites are the noncatalytic ones. The analogue could be displaced by excess ADP also from these sites; however, in contrast to the catalytic sites, no promotive effect was observed here. The obvious changes in the nucleotide binding behavior of the non-catalytic sites after depletion of endogenous nucleotides will be discussed.
Weber
J
J
Institut für Biochemie, Medizinische Universität zu Lübeck, Federal Republic of Germany.
Schmitt
S
S
Grell
E
E
Schäfer
G
G
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D005456:Fluorescent Dyes; D007290:Inosine Diphosphate / Q000494:pharmacology; D007293:Inosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009711:Nucleotides; D007293:Inosine Triphosphate; C055520:linear-benzoadenosine diphosphate; D000244:Adenosine Diphosphate; D007290:Inosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases