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

Phillip H Kuo, Robert K. Nakamoto

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 Δβ(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 γ subunit in multi-site, co-operative catalysis.

Original languageEnglish (US)
Pages (from-to)797-805
Number of pages9
JournalBiochemical Journal
Volume347
Issue number3
DOIs
StatePublished - May 1 2000
Externally publishedYes

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Escherichia coli
Protons
Adenosine Triphosphate
Mutation
Amino Acid Substitution
Catalysis
Cysteine
Conformations
Hydrolysis
Substitution reactions
Amino Acids

Keywords

  • Coupling
  • Isokinetic analysis
  • Rotational catalysis
  • Transition state

ASJC Scopus subject areas

  • Biochemistry

Cite this

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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 Δβ(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 γ subunit in multi-site, co-operative catalysis.",
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T2 - Functional interactions between residues in the proton transport site

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AU - Nakamoto, Robert K.

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N2 - 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 Δβ(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 γ subunit in multi-site, co-operative catalysis.

AB - 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 Δβ(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 γ subunit in multi-site, co-operative catalysis.

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