J. Biol. Chem., Vol. 262, Issue 7, 3146-3153, Mar, 1987
Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex
M Ronjat, JJ Lacapere, JP Dufour and Y Dupont
The plasma membrane of yeasts contains an H+-ATPase similar to the other
cation transport ATPases of eukaryotic organisms. This enzyme has been
purified and shows H+ transport in reconstituted vesicles. In the presence
of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+- ATPase and
supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP
(Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP
for ATP hydrolysis indicates that terbium-associated nucleotides bind to
the catalytic site of the H+-ATPase. The fluorescent properties of the
Tb-FTP complex were used to study the active site of the H+-ATPase.
Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide
site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP,
and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0,
and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the
presence of an excess of Tb3+ with an activation constant Ka = 25 microM
for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+
binding are independent entities. In standard conditions these sites would
be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an
important regulatory role of divalent cations on the activity of H+-ATPase.
Replacement of H2O by D2O in the medium suggests the existence of two types
of nucleotide binding sites differing by the hydration state of the Tb3+
ion in the bound Tb-FTP complex.
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