Covalent modification of the catalytic sites of the H +-ATPase from chloroplasts with 2-nitreno-ADP. Modification of the catalytic site 1 (tight) and catalytic sites 1 and 2 together impairs both uni-site and multi-site catalysis of ATP synthesis and ATP hydrolysis

Abstract

After isolation and purification, the H +-ATPase from chloroplasts, CF 0F 1, contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[α- 32P]ADP leads to tight binding of azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[α- 32P]ADP, the covalently bound label was found exclusively at β-Tyr-362. Incubation conditions with 2-azido-[α- 32P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. For measurements of the degree of inhibition by covalent modification, CF 0F 1 was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K +/valinomycin diffusion potential. The rate of ATP synthesis was 50–80 s −1, and the rate of ATP hydrolysis was 15 s −1 measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF 0F 1. Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF 0F 1. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with derivatised CF 0F 1 and with non-derivatised CF 0F 1. ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.