The three catalytic sites of the FoF1 ATP synthase interact through a cooperative mechanism that is required for the promotion of catalysis. Replacement of the conserved α subunit Arg-376 in the Escherichia coli F1 catalytic site with Ala or Lys resulted in turnover rates of ATP hydrolysis that were 2 x 103-fold lower than that of the wild type. Mutant enzymes catalyzed hydrolysis at a single site with kinetics similar to that of the wild type; however, addition of excess ATP did not chase bound ATP, ADP, or Pi from the catalytic site, indicating that binding of ATP to the second and third sites failed to promote release of products from the first site. Direct monitoring of nucleotide binding in the αR376A and αR376K mutant F1 by a tryptophan in place of βTyr-331 (Weber et al. (1993) J. Biol, Chem. 268, 20126-20133) showed that the catalytic sites of the mutant enzymes, like the wild type, have different affinities and therefore, are structurally asymmetric. These results indicate that αArg-376, which is close to the β- or γ-phosphate group of bound ADP or ATP, respectively, does not make a significant contribution to the catalytic reaction, but coordination of the arginine to nucleotide filling the low-affinity sites is essential for promotion of rotational catalysis to steady-state turnover.
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