Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1).
The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.
Günther
S
S
ITZ, School of Veterinary Medicine, Hannover, Germany.
Huchzermeyer
B
B
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000535:Aluminum / Q000494:pharmacology; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D005461:Fluorine / Q000494:pharmacology; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D013053:Spectrophotometry; D018724:Spinacia oleracea / Q000737:chemistry*
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009711:Nucleotides; C053629:fluoroaluminum; D005461:Fluorine; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000535:Aluminum; D006180:Proton-Translocating ATPases