Covalent modification of the non-catalytic sites of the H+-ATPase from chloroplasts with 2-azido-[alpha-P-32]ATP and its effect on ATP synthesis and ATP hydrolysis

Incubation of the isolated H+-ATPase from chloroplasts, CF0F1, with 2-azido -[alpha-P-32]ATP leads to the binding of this nucleotide to different sites . These sites were identified after removal of free nucleotides, UV-irradia tion and trypsin treatment by separation of the tryptic peptides by ion exc hange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines we re used to distinguish between catalytic (beta -Tyr362) and non-catalytic ( beta -Tyr385) sites. Several incubation procedures were developed which all ow a selective occupation of each of the three noncatalytic sites. The non- catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 muM 2-azido-[alpha-P-32]ATP, that with the intermed iate dissociation constant (site 5) at 2 muM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a rep lacement by 2-azido-[alpha-P-32]ATP was possible. CF0F1 with non-covalently bound 2-azido-[alpha-P-32]ATP and after covalent derivatization was recons tituted into liposomes and the rates of ATP synthesis as well as ATP hydrol ysis were measured after energization of the proteoliposomes by Delta pH/De lta . Noncovalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being p roportional to the degree of derivatization. Extrapolation to complete inhi bition indicates that derivatization of one site (either 4 or 5 or 6) is su fficient to block completely multi-site catalysis. The rates of ATP synthes is and ATP hydrolysis were measured as a function of the ADP and ATP concen tration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF0F1. Uni-site ATP synthesis and ATP hydrolysis were n ot inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that mult i-site catalysis requires some flexibility between beta- and a-subunits whi ch is abolished by covalent derivatization of beta -Tyr385 with a 2-nitreno -adenine nucleotide. Conformational changes connected with energy transduct ion between the F-0-part and the F-1-part are either not required for uni-s ite ATP synthesis or they are not impaired by the derivatization of any of the three beta -Tyr385. (C) 2001 Elsevier Science B.V. All rights reserved.