Mutational analysis of the nucleotide binding sites of the yeast vacuolar proton-translocating ATPase.
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 beta 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. 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.
MacLeod
K J
KJ
Departments of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Vasilyeva
E
E
Baleja
J D
JD
Forgac
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014617:Vacuoles / Q000201:enzymology*
D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases