Kinetic analysis of cooperative interactions induced by Mn sup 2+ binding to the chloroplast H sup + -ATPase

The kinetics of Mn{sup 2+} binding to three cooperatively interacting sites in chloroplast H{sup +}-ATPase (CF{sub 1}) were measured by EPR following rapid mixing of the enzyme with MnCl{sub 2} with a time resolution of 8 ms. Mixing of the enzyme-bound Mn{sup 2+} with MgCl{sub 2} 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 CF{sub 1}, the binding to all three sites had similar on-rate constants of (1.1 {plus minus} 0.04) {times} 10{sup 4} M{sup {minus}1}s{sup {minus}1}. (2) Site segregation was found in the release of ions with off-rate constants of 0.69 {plus minus} 0.04 s{sup {minus}1} for the first two and 0.055 {plus minus} 0.003 s{sup {minus}1} for the third. (3) Addition of one ADP per CF{sub 1} caused a decrease in the off-rate constants. (4) Heat activation of CF{sub 1} increased the on-rate constant to (4.2 {plus minus} 0.92) {times} 10{sup 4} M{sup {minus}1} s{sup {minus}1} and the off-rate constants of the first two and the third site to 1.34 {plus minus} 0.08 and 0.16 {plus minus} 0.07 s{sup {minus}1}, respectively. (5) The calculated thermodynamic dissociation constantsmore » 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{sup +}-ATPase. The data support the suggestion that regulation induces sequential progress of catalysis through the three active sites of the enzyme.« less