title: E. coli F1-ATPase: site-directed mutagenesis of the beta-subunit.
abstract: Residues beta Glu-181 and beta Glu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP. Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1. In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity. Residues beta Gly-149 and beta Gly-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 beta-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 beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002455:Cell Division; D002462:Cell Membrane / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D010710:Phosphates; D004024:Dicyclohexylcarbodiimide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases