Covalent modification of the catalytic sites of the H+-ATPase from chloroplasts, CF0F1, with 2-azido-[alpha-P-32]ADP: modification of the catalytic site 2 (loose) and the catalytic site 3 (open) impairs multi-site, but not uni-site catalysis of both ATP synthesis and ATP hydrolysis

The H+-ATPase from chloroplasts, CF0F1, was isolated and purified. The enzy me contained one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-P-32]AD(T)P leads t o a tight binding of the azido-nucleotides. Free nucleotides were removed b y three consecutive passages through centrifugation columns, and after UV-i rradiation, the label was covalently bound. The labelled enzyme was digeste d by trypsin, the peptides were separated by ion exchange chromatography in to nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these we re then separated by reversed phase chromatography. Amino acid sequence ana lysis was used to identify the type of the nucleotide binding site. After i ncubation with 2-azido-[alpha-P-32]ADP, the covalently bound label was foun d exclusively at beta-Tyr-362, i.e. binding occurs only to catalytic sites. Incubation conditions with 2-azido-[alpha-P-32]ADP were varied, and condit ions were found which allow selective binding of the label to different cat alytic sites, either to catalytic site 2 or to catalytic site 3. For measur ements of the degree of inhibition by covalent modification, CF0F1 was reco nstituted into phosphatidylcholine liposomes, and the membranes were energi sed by an acid-base transition in the presence of a K+/valinomycin diffusio n potential. The rate of ATP synthesis was 120 s(-1), and the rate of ATP h ydrolysis was 20 s(-1), both measured under multi-site conditions. Covalent modification of either catalytic site 2 or catalytic site 3 inhibited both ATP synthesis and ATP hydrolysis, the degree of inhibition being proportio nal to the degree of modification. Extrapolation to complete inhibition ind icates that modification of one catalytic site, either site 2 or site 3, is sufficient to completely block multi-site ATP synthesis and ATP hydrolysis . 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 cond itions with covalently modified CF0F1 and with nonmodified CF0F1. The resul t was that uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent modification of either catalytic site 2 or site 3. The results ind icate cooperative interactions between catalytic nucleotide binding sites d uring multi-site catalysis, whereas neither uni-site ATP synthesis nor uni- site ATP hydrolysis require interaction with other sites. (C) 2000 Elsevier Science B.V. All rights reserved.