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

After isolation and purification, the Hi-ATPase from chloroplasts, CF0F1, c ontains one endogenous ADP at a catalytic site, and two endogenous ATP at n on-catalytic sites. Incubation with 2-azido-[alpha-P-32]ADP leads to tight binding of azido-nucleotides. Free nucleotides were removed by three consec utive passages through centrifugation columns, and upon UV-irradiation most of the label was covalently bound. The labelled enzyme was digested by try psin, the peptides were separated by ion exchange chromatography into nitre no-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis wa s used to identify the type of the nucleotide binding site. After incubatio n with 2-azido-[alpha-P-32]ADP, the covalently bound label was found exclus ively at beta-Tyr-362. Incubation conditions with 2-azido-[alpha-P-32]ADP w ere 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 de creasing 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, CF0F1 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 synth esis 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 hydr olysis equally, the degree of inhibition being proportional to the degree o f modification. Extrapolation to complete inhibition indicates that derivat isation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitre no-ADP is bound per mol CF0F1. Derivatisation of catalytic sites 1 and 2 to gether extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bou nd per CF0F1. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to u ni-site conditions with derivatised CF0F1 and with non-derivatised CF0F1. A TP synthesis and ATP hydrolysis under uni-site and under multi-site conditi on were inhibited by covalent modification of either catalytic site 1 or ca talytic 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. (C) 2000 Elsevier Scie nce B.V. All rights reserved.