PMID:10769185
| Citation |
Kuo, PH and Nakamoto, RK (2000) Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site. Biochem. J. 347 Pt 3:797-805 |
|---|---|
| Abstract |
Subunit a of the ATP synthase F(o) sector contains a transmembrane helix that interacts with subunit c and is critical for H(+) transport activity. From a cysteine scan in the region around the essential subunit a residue, Arg-210, we found that the replacement of aGly-213 greatly attenuated ATP hydrolysis, ATP-dependent proton pumping and Delta mu(H)+-dependent ATP synthesis. Various amino acid substitutions caused similar effects, suggesting that functional perturbations were caused by altering the environment or conformation of aArg-210. aG213N, which was particularly severe in effect, was suppressed by two second-site mutations, aL251V and cD61E. These mutations restored efficient coupling; the latter also increased ATP-dependent proton transport rates. These results were consistent with the proposed functional interaction between aArg-210 and cAsp-61, the likely carrier of the transported proton. From Arrhenius analysis of steady-state ATP hydrolytic activity, the transport mutants had large increases in the transition-state enthalpic and entropic parameters. Linear isokinetic relationships demonstrate that the transport mechanism is coupled to the rate-limiting catalytic transition-state step, which we have previously shown to involve the rotation of the gamma subunit in multi-site, co-operative catalysis. |
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| Keywords |
Adenosine Triphosphate/biosynthesis; Adenosine Triphosphate/metabolism; Amino Acid Substitution/genetics; Arginine/genetics; Arginine/metabolism; Binding Sites; Biological Transport, Active/drug effects; Blotting, Western; Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology; Catalysis/drug effects; Cysteine/genetics; Cysteine/metabolism; Electron Transport/drug effects; Escherichia coli/drug effects; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Genes, Suppressor/genetics; Intracellular Membranes/drug effects; Intracellular Membranes/enzymology; Kinetics; NAD/metabolism; Oxidative Phosphorylation/drug effects; Protein Structure, Secondary; Proton-Motive Force/drug effects; Proton-Translocating ATPases/chemistry; Proton-Translocating ATPases/genetics; Proton-Translocating ATPases/metabolism; Protons; Suppression, Genetic/genetics; Thermodynamics |
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