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Transport protons do not participate in ATP synthesis/hydrolysis at the nucleotide binding site of the H + ‐ATPase from chloroplasts
Author(s) -
Labahn Andreas,
Gräber Peter
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)81439-s
Subject(s) - atp hydrolysis , atpase , chemistry , nucleotide , photophosphorylation , thylakoid , chemiosmosis , dithiothreitol , enzyme , f atpase , atp synthase gamma subunit , hydrolysis , adenine nucleotide , atp synthase , chloroplast , biochemistry , stereochemistry , gene
The H + ‐ATPase from chloroplasts, CF 0 P 1 , was brought into the active, reduced state by illumination of thylakoids in the presence of thioredoxin and dithiothreitol. Uni‐site ATP synthesis was initiated by the addition of 20 nM [α‐ 32 P]ADP, and enzyme‐bound and free nucleotides were separated by a pressure column. The ratio of enzyme‐bound ADP to ATP was 0.55 ± 0.05. In a second experiment, uni‐site ATP hydrolysis under energized conditions was initiated by the addition of 36 nM [α‐ 32 P]ATP; enzyme‐bound and free nucleotides were separated by a pressure column. Both procedures were carried out under continuous illumination. The ratio of enzyme‐bound ADP to ATP was 0.46 ± 0.04. In a third experiment, uni‐site ATP hydrolysis under de‐energized conditions was initiated by the addition of 39 nM [α‐ 32 P]ATP and NH 4 Cl/valinomycin in the absence of illumination. Free and enzyme‐bound nucleotides were separated also by a pressure column. The ratio of enzyme‐bound ADP to ATP was 0.43 ± 0.02. This ratio was always the same irrespective of whether the reaction runs in the synthesis or the hydrolysis direction. Furthermore, the ratio does not depend on the membrane energization. We conclude, therefore, that the protons are not directly involved in the reaction at the catalytic site.