The a Subunit ala-217 → arg Substitution Affects Catalytic Activity of F1F0 ATP Synthase

A large number of mutations affecting the F0 sector of Escherichia coli F1F0 ATP synthase have been constructed and characterized. A subset of the missense mutations resulted in fully assembled enzyme complexes blocked in proton translocation and displaying marked decreases in ATP hydrolysis activity. The catalytic activities of one such mutant enzyme, aala-217→arg, have been determined using both multisite and unisite catalysis conditions. As expected, the Vmax of the aala-217→arg enzyme was reduced under conditions of saturating substrate concentration. However, the F0 sector amino acid substitution did not affect nucleotide occupancy of the noncatalytic sites. Moreover, the microscopic rate constants measured using unisite methods yielded no significant differences between the intact wild type F1F0 ATP synthase and the aala-217→arg mutant enzyme. In general, the values for unisite activities in both preparations were very similar to numbers reported in the literature for E. coli F1-ATPase. The results suggest that the aala-217→arg substitution resulted in a defect in catalytic cooperativity and most likely altered the enzyme by inhibiting the rotational mechanism of F1F0 ATP synthase.