Twenty-two mutants between βGlu-161 and βLys-201 of Escherichia coli H+-ATPase ft subunit could grow by oxidative phosphorylation, but 11 other such mutants, βGlu-181-> Gin, Asp, Asn, Thr, Ser, Ala, or Lys and βArg-182->Lys, Ala, Glu, or Gin, could not. The βAsp-181, βLys-182, and other defective mutants had 1.4, 1, and < 0.1%, respectively, of the wild-type membrane ATPase activity. Partially purified F, -ATPases from all mutants at positions 181 and 182, except for the βAsp-181 and βLys-182 mutants, showed very low unisite catalysis. Purified F1-ATPases of the βGln-181 and βAla-181 mutants showed no multisite (or steady state) catalysis and slow unisite catalysis (≤1% of that of the wild type): their defects could be attributed to decreased catalytic rates (low k+2 and k-2). Changes of the k+2 and k-2 values in the βAsp-181 enzyme, which showed detectable multi-and unisite catalysis, were less marked (27 and 21%, respectively, of wild-type rates). The βGln-182 enzyme showed defective catalysis (≤0.1% of the multi- and∼1% of the unisite catalyses of the wild type), whereas the βLys-182 enzyme showed 1 and 85% of the wild-type multisite and unisite catalytic rates, respectively. βLys-182 had wild-type values of k+2 and k_2, but βGln-182 had k+2 about 10-fold lower than that of wild type. The position 181 and 182 mutant enzymes had significantly increased Kd (k-1/k+1) values, reflecting decreased substrate binding. These results suggest that βGlu-181 and βArg-182 are essential for substrate binding, although mutations with conservative substitutions at these positions do not have drastic effects. This study also indicates the importance of the conserved Gly-Glu-Arg (GER) sequence (β180-β182).
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Park et al. (1994) studied this question.