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Vol. 273, Issue 1, 150-156, January 2, 1998
From the Departments of Cellular and Molecular Physiology and
To further define the structure of the nucleotide
binding sites on the vacuolar proton-translocating ATPase
(V-ATPase), the role of aromatic residues at the catalytic sites
was probed using site-directed mutagenesis of the VMA1 gene
that encodes the A subunit in yeast. Substitutions were made at three
positions (Phe452, Tyr532, and
Phe538) that correspond to residues observed in the crystal
structure of the homologous Site-directed mutations were also made at residues (Phe479
and Arg483) that are postulated to be contributed by the A
subunit to the noncatalytic nucleotide binding sites. Generally,
substitutions at these positions led to decreases in activity ranging
from 30 to 70% relative to wild type as well as modest decreases in
Km for ATP. Interestingly, the R483E and R483Q
mutants showed a time-dependent increase in ATPase activity
following addition of ATP, suggesting that events at the noncatalytic
sites may modulate the catalytic activity of the enzyme.
Mutational Analysis of the Nucleotide Binding Sites of the Yeast
Vacuolar Proton-translocating ATPase
, and Biochemistry, Tufts University School of Medicine,
Boston, Massachusetts 02111
subunit of the bovine mitochondrial
F-ATPase to be in proximity to the adenine ring of bound ATP. Although conservative substitutions at these positions had relatively little effect on V-ATPase activity, replacement with nonaromatic residues (such as alanine or serine) caused either a complete loss of activity (F452A) or a decrease in the affinity for ATP (Y532S and F538A). The
F452A mutation also appeared to reduce stability of the V-ATPase complex. These results suggest that aromatic or hydrophobic residues at
these positions are essential to maintain activity and/or high affinity
binding to the catalytic sites of the V-ATPase.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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