Tyr-341 of the β Subunit Is a Major K_m-Determining Residue of TF₁-ATPase: Parallel Effect of Its Mutations on K_d(ATP) of the β Subunit and on K_m(ATP) of the α₃β₃ γ Complex

Abstract

Residue Tyr-341 of the F₁-ATPase β subunit from a thermophilic Bacillus strain, PS3, was mutagenized to leucine, cysteine or alanine. Each of the mutated β subunits was isolated and its affinity for ATP-Mg was examined by means of difference circular dichroism and differential titration calorimetry. The K_d values for ATP-Mg obtained were: βY341 (wild type), 0.015mM; βY341L, 0.7mM; βY341C and βY341A, >3mM. All the mutant β subunits could be reconstituted into the α₃β₃γ complex with α and γ subunits. The α₃β_(mutant)3γ complexes hydrolyzed ATP with apparent V_max values larger than that of the α₃β_(WILD)3γ complex. The apparent Km values of the α₃β_(mutan)3γ complexes increased in parallel with the K_d values for ATP-Mg of the isolated mutant β subunits. These results indicate that residue βY341 is directly involved in the catalytic ATP-Mg binding and is a major K_m-determining residue of F₁-ATPase.