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E. coli F 1 ‐ATPase: Site‐directed mutagenesis of the β‐subunit
Author(s) -
Parsonage Derek,
Wilke-Mounts Susan,
Senior Alan E.
Publication year - 1988
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(88)80397-1
Subject(s) - mutant , protein subunit , mutagenesis , beta (programming language) , atpase , cooperativity , biochemistry , chemistry , site directed mutagenesis , pi , microbiology and biotechnology , biology , enzyme , gene , computer science , programming language
Residues βGlu‐181 and βGlu‐192 of E. coli F 1 ‐ATPase (the DCCD‐reactive residues) were mutated to Gln. Purified βGln‐181 F 1 showed 7‐fold impairment of ‘unisite’ P i formation from ATP and a large decrease in affinity for ATP. Thus the β‐181 carboxyl group in normal F 1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 × 10 4 ‐ to 548‐fold in βGln‐181 F 1 . In contrast, purified βGln‐192 F 1 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 F 1 ‐ATPase ranged from 5 to 61%, with the double mutant F 1 less impaired than either single mutant. F 1 preparations containing βIle‐154 showed 2‐fold activation after release from membranes, suggesting association with F 0 restrained turnover on F 1 in these mutants.