Kinetic analysis of cooperative interactions induced by Mn2+ binding to the chloroplast H(+)-ATPase.
The kinetics of Mn2+ binding to three cooperatively interacting sites in chloroplast H(+)-ATPase (CF1) were measured by EPR following rapid mixing of the enzyme with MnCl2 with a time resolution of 8 ms. Mixing of the enzyme-bound Mn2+ with MgCl2 gave a measure of the rate of exchange. The data could be best fitted to a kinetic model assuming three sequential, positively cooperative binding sites. (1) In the latent CF1, the binding to all three sites had a similar on-rate constants of (1.1 +/- 0.04) X 10(4) M-1s-1. (2) Site segregation was found in the release of ions with off-rate constants of 0.69 +/- 0.04 s-1 for the first two and 0.055 +/- 0.003 s-1 for the third. (3) Addition of one ADP per CF1 caused a decrease in the off-rate constants to 0.31 +/- 0.02 and 0.033 +/- 0.008 s-1 for the first two and the third sites, respectively. (4) Heat activation of CF1 increased the on-rate constant to (4.2 +/- 0.92) X 10(4) M-1s-1 and the off-rate constants of the first two and the third site to 1.34 +/- 0.08 and 0.16 +/- 0.07 s-1, respectively. (5) The calculated thermodynamic dissociation constants were similar to those previously obtained from equilibrium binding studies. These findings were correlated to the rate constants obtained from studies of the catalysis and regulation of the H(+)-ATPase. The data support the suggestion that regulation induces sequential progress of catalysis through the three active sites of the enzyme.
Hiller
R
R
Department of Biochemistry, Tel Aviv University, Israel.
Carmeli
C
C
D001665:Binding Sites; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006358:Hot Temperature; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D010940:Plant Proteins; D008345:Manganese; D006180:Proton-Translocating ATPases