CONSERVED GLU-181 AND ARG-182 RESIDUES OF ESCHERICHIA-COLI H+-ATPASE (ATP SYNTHASE) BETA-SUBUNIT ARE ESSENTIAL FOR CATALYSIS - PROPERTIES OF 33 MUTANTS BETWEEN BETA-GLU-181 AND BETA-LYS-801 RESIDUES

Twenty-two mutants between beta Glu-161 and beta Lys-201 of Escherichia coli H+-ATPase beta subunit could grow by oxidative phosphorylation, but 11 other such mutants, beta Glu-181-->Gln, Asp, Asn, Thr, Ser, Ale, or Lys and beta Arg-182-->Lys, Ala, Glu, or Gin, could not. The beta Asp-181, beta Lys-182, and other defective mutants had 1.4, 1, and < 0.1%, respectively, of the wild-type membrane ATPase activity. Partially purified F-1-ATPases from all mutants at positions 181 and 182, except for the beta Asp-181 and beta Lys-182 mutants, showed very low unisite catalysis. Purified F-1-ATPases of the beta Gln-181 and beta Ala-181 mutants showed no multisite (or steady state) catalysis and slow unisite catalysis (less than or equal to 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 beta Asp-181 enzyme, which showed detectable multi- and unisite catalysis, were less marked (27 and 21%, respectively, of wild-type rates). The beta Gln-182 enzyme showed defective catalysis (less than or equal to 0.1% of the multi- and similar to 1% of the unisite catalyses of the wild type), whereas the beta Lys-182 enzyme showed 1 and 85% of the wild-type multisite and unisite catalytic rates, respectively. beta Lys-182 had wild-type values of k(+2) and k(-2), but beta Gln-182 had k(+2) about 10-fold lower than that of wild type. The position 181 and 182 mutant enzymes had significantly increased K-d (k(-1)/k(+1)) values, reflecting decreased substrate binding. These results suggest that beta Glu-181 and beta Arg-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 (beta 180-beta 182).