Amino acid replacements at seven different histidines in the yeast plasma membrane H+-ATPase reveal critical positions at His285 and His701

The histidine residues in the plasma membrane H+-ATPase (Pma1) of the yeast Saccharomyces cerevisiae that are involved in membrane activity were identified by combining systematic site-directed mutagenesis and kinetic analysis. Only substitutions at H701 with the amino acids Asp, Gln and Arg produced a lethal effect, indicating that H701 is involved in H+-ATPase activity. Mutations at H285Q and H285R had no effect on the cells in normal growth conditions, but growth of H285Q was sensitive to acid pH. Both also exhibited optimal ATPase-specific activity and other kinetic parameters at a substrate concentration much lower than the maximum enzyme concentration. It is suggested that H285Q is involved in the transition step of the ATP hydrolysis catalytic cycle.