ATP-hydrolysis in chloroplasts: Uni-site 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.