Covalent modification of the non-catalytic sites of the H(+)-ATPase from chloroplasts with 2-azido-[alpha-(32)P]ATP and its effect on ATP synthesis and ATP hydrolysis.
Incubation of the isolated H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange 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 were used to distinguish between catalytic (beta-Tyr362) and non-catalytic (beta-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 microM 2-azido-[alpha-(32)P]ATP, that with the intermediate dissociation constant (site 5) at 2 microM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[alpha-(32)P]ATP was possible. CF(0)F(1) with non-covalently bound 2-azido-[alpha-(32)P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by Delta pH/Delta phi. Non-covalent 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 proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF(0)F(1). Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between beta- and alpha-subunits which is abolished by covalent derivatization of beta-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F(0)-part and the F(1)-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three beta-Tyr385.
Possmayer
F E
FE
Institut für Physikalische Chemie, Universität Freiburg, Germany.
Hartog
A F
AF
Berden
J A
JA
Gräber
P
P
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000345:Affinity Labels / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D007700:Kinetics; D008956:Models, Chemical; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D000244:Adenosine Diphosphate; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases