TYR-341 OF THE BETA-SUBUNIT IS A MAJOR K-M-DETERMINING RESIDUE OF TF1-ATPASE - PARALLEL EFFECT OF ITS MUTATIONS ON K-D((ATP)) OF THE BETA-SUBUNIT AND ON K-M((ATP)) OF THE ALPHA(3)BETA(3)GAMMA COMPLEX

Residue Tyr-341 of the F-1-ATPase beta subunit from a thermophilic Bac illus strain, PS3, was mutagenized to leucine, cysteine or alanine. Ea ch of the mutated beta subunits was isolated and its affinity for ATP- Mg was examined by means of difference circular dichroism and differen tial titration calorimetry. The K-d values for ATP-Mg obtained were: b eta Y341 (wild type), 0.015mM; beta Y341L, 0.7mM; beta Y341C and beta Y341A, >3mM. All the mutant beta subunits could be reconstituted into the alpha(3) beta(3) gamma complex with alpha and gamma subunits. The alpha(3) beta((mutant)3) gamma complexes hydrolyzed ATP with apparent V-max values larger than that of the alpha(3) beta(wild)(3) gamma comp lex. The apparent K-m values of the alpha(3) beta((mutant)3) gamma com plexes increased in parallel with the K-d values for ATP-Mg of the iso lated mutant beta subunits. These results indicate that residue beta Y 341 is directly involved in the catalytic ATP-Mg binding and is a majo r K-m-determining residue of F-1-ATPase.