Inactivation of chloroplast H(+)-ATPase by modification of Lys beta 359, Lys alpha 176 and Lys alpha 266.
Treatment of isolated, latent chloroplast ATPase with pyridoxal-5-phosphate (pyridoxal-P) in presence of Mg2+ causes inhibition of dithiothreitol-activated plus heat-activated ATP hydrolysis. The amount of [3H]pyridoxal-P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 +/- 1 pyridoxal-P/enzyme, almost equally distributed between the alpha- and beta-subunits. Inactivation, however, is complete after binding of 1.5-2 pyridoxal-P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H-labeled pyridoxal-P into beta- and alpha-subunits. Phosphate prevents labeling of the alpha-subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the alpha- and beta-subunits. Cleavage of the pyridoxal-P-labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lys beta 359, Lys alpha 176 and Lys alpha 266, which are closely related to proposed nucleotide-binding regions of the alpha- and beta-subunits.
Horbach
M
M
Institut für Biochemie der Pflanzen, Heinrich Heine Universität Düsseldorf, Federal Republic of Germany.
Meyer
H E
HE
Bickel-Sandkötter
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006868:Hydrolysis; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000235:genetics; D011732:Pyridoxal Phosphate / Q000378:metabolism
D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D008239:Lysine; D003488:Cyanogen Bromide