ATP-HYDROLYSIS IN CHLOROPLASTS - UNISITE CATALYSIS AND EVIDENCE FOR HETEROGENEITY OF CATALYTIC SITES

ATP-hydrolysis was measured with thylakoids under uni-site conditions. The rate constant for the ATP binding is 106 M-1 · s-1, the 'equilibrium constant' of the enzyme-substrate complex, EADPP1 EATP, is 0.4. The rate constants for the P1-release and the ADP-release are 0.23 s-1 and 0.07 s-1. This indicates that the enzyme carries out a complete turnover under uni-site conditions. The interaction of the nucleotide binding sites was investigated by first measuring the [γ-32P]ATP hydrolysis under uni-site conditions. After that, 1 mM unlabeled ATP was added, so that all ATP-binding sites were occupied. The [γ-32P]ATP, bound to the first site, was hydrolyzed at a rate of 0.8 ATP (CF0F1 s). In a second experiment, first unlabeled ATP was hydrolyzed under uni-site conditions, and then 1 mM [γ-32P]ATP was added. Under otherwise identical conditions, this allows the measurement of the rate of ATP hydrolysis catalyzed by the second (and possibly third) site. A rate of 40 ATP (CF0F1 s) was found. It was concluded that there exists a heterogeneity of the ATP-hydrolyzing sites on CF0F1 in thylakoid membranes. © 1990.