Tyr-341 of the β Subunit Is a Major Km-Determining Residue of TF1-ATPase: Parallel Effect of Its Mutations on Kd(ATP) of the β Subunit and on Km(ATP) of the α3β3γ Complex1

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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 Kd values for ATP-Mg obtained were: βY341 (wild type), 0.015 mM; βγ341L, 0.7 mM; βγ341C and βγ341A, >3mM. All the mutant β subunits could be reconstituted into the α3β3γ complex with a and y subunits. The α3β(mutant) γ complexes hydrolyzed ATP with apparent Vmax values larger than that of the α3β(WILD)3 γ complex. The apparent Km values of the α3β(mutant)3γ complexes increased in parallel with the Kd values for ATP-Mg of the isolated mutant β subunits. These results indicate that residue βγ341 is directly involved in the catalytic ATP-Mg binding and is a major Km -determining residue of F1-ATPase.

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