The role of H211 of the yeast F1-ATPase beta-subunit was investigated by site-specific mutagenesis and characterization of the resulting enzymes. Five amino acids (N, D, I, K, and A) were substituted for H211 of the ATP2 gene. The mutated genes were expressed in an atp2::LEU2 host, and only yeast expressing H211N respired aerobically. The respiratory phenotypes of the other four mutants were suppressed by a second site mutation (L203F). The ATPases from the single mutant strains were unstable when removed from the mitochondrial inner membrane, preventing purification. Submitochondrial particles were prepared from each strain and the activities were stable under a variety of conditions, allowing determination of Vmax and Km for ATP hydrolysis. Mutations of H211 caused increases in Km of 3.7- to 7.4-fold, while L203F had little effect. The suppressive effect of the L203F mutation was also expressed in the Km values of the double mutant strains. The ATPases from the H211 mutants had diminished sensitivity to oligomycin, and their pH optima were 1.5-2.0 units less than the wild-type optimum. Values of pKa for the groups involved in catalysis were estimated for the wild-type enzyme and three H211 mutants (N, D, and K). Each mutant enzyme showed a substantial decrease in the pKa of the group(s) which serves as a base in acid-base catalysis. The results of this study demonstrate that H211 is important in maintaining the structure of the wild-type enzyme complex and also contributes to the structure of the active site. L203 is also required for the stability of the enzyme complex and may have a structural or functional interaction with H211. Neither H211 nor L203 is required for catalysis by F1.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|