E. coli F1 -ATPase: Site-directed mutagenesis of the β-subunit

AbstractResidues βGlu-181 and βGlu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified βGln-181 F1 showed 7-fold impairment of ‘unisite’ Pi formation from ATP and a large decrease in affinity for ATP. Thus the β-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 × 104- to 548-fold in βGln-181 F1. In contrast, purified βGln-192 F1 showed only 6-fold reduction in ‘multisite’ ATPase activity. Residues βGly-149 and βGly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative ‘flexible loop’ in β-subunit. Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing βIle-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants