title: The a subunit ala-217 --> arg substitution affects catalytic activity of F(1)F(0) ATP synthase.
abstract: A large number of mutations affecting the F(0) sector of Escherichia coli F(1)F(0) 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, a(ala-217-->arg), have been determined using both multisite and unisite catalysis conditions. As expected, the V(max) of the a(ala-217-->arg) enzyme was reduced under conditions of saturating substrate concentration. However, the F(0) 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 F(1)F(0) ATP synthase and the a(ala-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 F(1)-ATPase. The results suggest that the a(ala-217-->arg) substitution resulted in a defect in catalytic cooperativity and most likely altered the enzyme by inhibiting the rotational mechanism of F(1)F(0) ATP synthase.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Gardner
authors.forename: B D
authors.initials: BD
authors.lastname: Cain
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000409:Alanine / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry*; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D016296:Mutagenesis; D020125:Mutation, Missense; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000255:Adenosine Triphosphate; D001120:Arginine; D006180:Proton-Translocating ATPases; D000409:Alanine