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.
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.
Kuo
P H
PH
University of Virginia, Department of Molecular Physiology and Biological Physics, P.O. Box 10011, Charlottesville, VA 22906-0011, USA.
Nakamoto
R K
RK
D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics*; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D015153:Blotting, Western; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016153:Genes, Suppressor / Q000235:genetics; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects; D017433:Protein Structure, Secondary; D018892:Proton-Motive Force / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011522:Protons; D013489:Suppression, Genetic / Q000235:genetics*; D013816:Thermodynamics
D011522:Protons; D009243:NAD; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; D001120:Arginine; D006180:Proton-Translocating ATPases; D003545:Cysteine