Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F₁-ATPase β subunit of Escherichia coli

βLys-155 in the glycine-rich sequence of the β subunit of Escherichia coli F₁-ATPase has been shown to be near the γ-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of βLys- 155 and βThr-156, mutants (βLys-155 → Ala, Ser, or Thr; βThr-156 → Ala, Cys, Asp, or Ser; βLys-155/βThr-156 → βThr-155/βLys-156; and βThr- 156/βVal-157 → βAla-156/βThr-157) were constructed, and their properties were studied extensively. The βSer-156 mutant was active in ATP synthesis and had ~1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had <0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F₁ in their membranes as the wild type. Purified mutant enzymes (βAla- 155, βSer-155, βAla-156, and βCys-156) showed low rates of multisite (<0.02% of the wild type) and unisite (<1.5% of the wild type) catalyses. The k₁ values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the βAla-156 and βCys-156 enzymes and 10²-fold lower with the βAla-155 and βSer-155 enzymes. The βThr-156 → Ala or Cys enzyme showed an altered response to Mg²⁺, suggesting that βThr-156 may be closely related to Mg²⁺ binding. These results suggest that βLys-155 and βThr-156 are essential for catalysis and are possibly located in the catalytic site, although βThr-156 could be replaced by a serine residue.