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 mutage nesis of the VMA1 gene that encodes the A subunit in yeast, Substituti ons were made at three positions (Phe(452), Tyr(532), and Phe(538)) th at correspond to residues observed in the crystal structure of the hom ologous beta subunit of the bovine mitochondrial F-ATPase to be in pro ximity to the adenine ring of bound ATP. Although conservative substit utions at these positions had relatively little effect on V-ATPase act ivity, replacement with nonaromatic residues (such as alanine or serin e) caused either a complete loss of activity (F452A) or a decrease in the affinity for ATP (Y532S and F538A). The F452A mutation also appear ed to reduce stability of the V-ATPase complex. These results suggest that aromatic or hydrophobic res residues at these positions are essen tial to maintain activity and/or high affinity binding to the catalyti c sites of the V-ATPase. Site-directed mutations were also made at res idues (Phe(479) and Arg(483)) 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 rangin g from 30 to 70% relative to wild type as well as modest decreases in K-m for ATP, Interestingly, the R483E and R483Q mutants showed a time dependent increase in ATPase activity following addition of ATP, sugge sting that events at the noncatalytic sites may modulate the catalytic activity of the enzyme.