The Escherichia coli F(O)F1 γM23K uncoupling mutant has a higher K0.5 for P(i). Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway

Marwan K. Al-Shawi, Christian J. Ketchum, Robert K. Nakamoto

Research output: Contribution to journalArticle

Abstract

The Escherichia coli F(O)F1 ATP synthase uncoupling mutation, γM23K, was found to increase the energy of interaction between γ and β subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a P(i) release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and P(i) was determined for the γM23K F(O)F1. The K0.5 for ADP was not affected, but K0.5 for P(i) was approximately 7-fold higher even though the apparent V(max) was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 °C. During oxidative phosphorylation, the apparent dissociation constant (K(I)) for ATP was not affected and was 5-6 mM for both wild-type and γM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of Δμ(H)+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the γM23K F(O)F1 revealed that, like those of ATP hydrolysis, the transition state ΔH((+)) was much more positive and TΔS((+)) was much less negative, adding up to little change in ΔG((+)). These results suggested that ATP synthesis is inefficient because of an extra bond between γ and β subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the β subunits changes conformation and affinity for nucleotide.

Original languageEnglish (US)
Pages (from-to)12961-12969
Number of pages9
JournalBiochemistry®
Volume36
Issue number42
DOIs
StatePublished - Oct 21 1997
Externally publishedYes

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Escherichia coli
Hydrolysis
Adenosine Triphosphate
Kinetics
Catalysis
Adenosine Diphosphate
Enzymes
Mutation
Oxidative Phosphorylation
Substrates
Binding energy
Catalytic Domain
Nucleotides
Conformations
Stroke
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

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The Escherichia coli F(O)F1 γM23K uncoupling mutant has a higher K0.5 for P(i). Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway. / Al-Shawi, Marwan K.; Ketchum, Christian J.; Nakamoto, Robert K.

In: Biochemistry®, Vol. 36, No. 42, 21.10.1997, p. 12961-12969.

Research output: Contribution to journalArticle

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abstract = "The Escherichia coli F(O)F1 ATP synthase uncoupling mutation, γM23K, was found to increase the energy of interaction between γ and β subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a P(i) release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and P(i) was determined for the γM23K F(O)F1. The K0.5 for ADP was not affected, but K0.5 for P(i) was approximately 7-fold higher even though the apparent V(max) was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 °C. During oxidative phosphorylation, the apparent dissociation constant (K(I)) for ATP was not affected and was 5-6 mM for both wild-type and γM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of Δμ(H)+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the γM23K F(O)F1 revealed that, like those of ATP hydrolysis, the transition state ΔH((+)) was much more positive and TΔS((+)) was much less negative, adding up to little change in ΔG((+)). These results suggested that ATP synthesis is inefficient because of an extra bond between γ and β subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the β subunits changes conformation and affinity for nucleotide.",
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T1 - The Escherichia coli F(O)F1 γM23K uncoupling mutant has a higher K0.5 for P(i). Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway

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N2 - The Escherichia coli F(O)F1 ATP synthase uncoupling mutation, γM23K, was found to increase the energy of interaction between γ and β subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a P(i) release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and P(i) was determined for the γM23K F(O)F1. The K0.5 for ADP was not affected, but K0.5 for P(i) was approximately 7-fold higher even though the apparent V(max) was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 °C. During oxidative phosphorylation, the apparent dissociation constant (K(I)) for ATP was not affected and was 5-6 mM for both wild-type and γM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of Δμ(H)+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the γM23K F(O)F1 revealed that, like those of ATP hydrolysis, the transition state ΔH((+)) was much more positive and TΔS((+)) was much less negative, adding up to little change in ΔG((+)). These results suggested that ATP synthesis is inefficient because of an extra bond between γ and β subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the β subunits changes conformation and affinity for nucleotide.

AB - The Escherichia coli F(O)F1 ATP synthase uncoupling mutation, γM23K, was found to increase the energy of interaction between γ and β subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a P(i) release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and P(i) was determined for the γM23K F(O)F1. The K0.5 for ADP was not affected, but K0.5 for P(i) was approximately 7-fold higher even though the apparent V(max) was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 °C. During oxidative phosphorylation, the apparent dissociation constant (K(I)) for ATP was not affected and was 5-6 mM for both wild-type and γM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of Δμ(H)+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the γM23K F(O)F1 revealed that, like those of ATP hydrolysis, the transition state ΔH((+)) was much more positive and TΔS((+)) was much less negative, adding up to little change in ΔG((+)). These results suggested that ATP synthesis is inefficient because of an extra bond between γ and β subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the β subunits changes conformation and affinity for nucleotide.

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