The a subunit ala-217 -> arg substitution affects catalytic activity of F1F0 ATP synthase

A large number of mutations affecting the F-0 sector of Escherichia coli F1 F0 ATP synthase have been constructed and characterized. A subset of the mi ssense mutations resulted in fully assembled enzyme complexes blocked in pr oton translocation and displaying marked decreases in ATP hydrolysis activi ty. The catalytic activities of one such mutant enzyme, a(ala.217-->arg), h ave been determined using both multisite and unisite catalysis conditions. As expected, the V-max of the a(ala.217-->arg) enzyme was reduced under con ditions of saturating substrate concentration. However, the F-0 sector amin o acid substitution did not affect nucleotide occupancy of the noncatalytic sites. Moreover, the microscopic rate constants measured using unisite met hods yielded no significant differences between the intact wild type F1F0 A TP synthase and the a(ala.217-->arg) mutant enzyme. In general, the values for unisite activities in both preparations were very similar to numbers re ported in the literature for E. coli F-1-ATPase. The results suggest that t he a(ala.217-->arg) substitution resulted in a defect in catalytic cooperat ivity and most likely altered the enzyme by inhibiting the rotational mecha nism of F1F0 ATP synthase. (C) 2000 Academic Press.