Multiple-step kinetic mechanism of DNA-independent ATP binding and hydrolysis by Escherichia coli replicative helicase DnaB protein

Quantitative analysis using the rapid quench-flow method

Surendran Rajendran, Maria J. Jezewska, Wlodzimierz Bujalowski

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39 Citations (Scopus)

Abstract

The kinetic mechanism of DNA-independent binding and hydrolysis of ATP by the E. coli replicative helicase DnaB protein has been quantitatively examined using the rapid quench-flow technique. Single-turnover studies of ATP hydrolysis, in a non-interacting active site of the helicase, indicate that bimolecular association of ATP with the site is followed by the reversible hydrolysis of nucleotide triphosphate and subsequent conformational transition of the enzyme-product complex. The simplest mechanism, which describes the data, is a three-step sequential process defined. The sequential character of the mechanism excludes conformational transitions of the DnaB helicase prior to ATP binding. Analysis of relaxation times and amplitudes of the reaction allowed us to estimate all rate and equilibrium constants of partial steps of the proposed mechanism. The intrinsic binding constant for the formation of the (H-ATP) complex is K(ATP) = (1.3 ± 0.5) x 105 M-1. The analysis of the data indicates that a part of the ATP binding energy originates from induced structural changes of the DnaB protein-ATP complex prior to ATP hydrolysis. The equilibrium constant of the chemical interconversion is K(H) = k2/k-2 ~ 2 while the subsequent conformational transition is characterized by K3 = k3/k-3 ~ 30. The low value of K(H) and the presence of the subsequent energetically favorable conformational step(s) strongly suggest that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release. The combined application of single and multiple-turnover approaches show that all six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. Binding of ATP to the DnaB hexamer is characterized by the negative cooperativity parameter σ = 0.25(±0.1). The negative cooperative interactions predominantly affect the ground state of the enzyme-ATP complex. The significance of these results for the mechanism of the free energy transduction of the DnaB helicase is discussed. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)773-795
Number of pages23
JournalJournal of Molecular Biology
Volume303
Issue number5
DOIs
StatePublished - Nov 10 2000

Fingerprint

DnaB Helicases
Hydrolysis
Adenosine Triphosphate
Escherichia coli
DNA
Proteins
Catalytic Domain
Enzymes
Nucleotides

Keywords

  • ATPases
  • DNA replication
  • Helicases
  • Protein-nucleotide interactions
  • Rapid quench-flow kinetics

ASJC Scopus subject areas

  • Virology

Cite this

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title = "Multiple-step kinetic mechanism of DNA-independent ATP binding and hydrolysis by Escherichia coli replicative helicase DnaB protein: Quantitative analysis using the rapid quench-flow method",
abstract = "The kinetic mechanism of DNA-independent binding and hydrolysis of ATP by the E. coli replicative helicase DnaB protein has been quantitatively examined using the rapid quench-flow technique. Single-turnover studies of ATP hydrolysis, in a non-interacting active site of the helicase, indicate that bimolecular association of ATP with the site is followed by the reversible hydrolysis of nucleotide triphosphate and subsequent conformational transition of the enzyme-product complex. The simplest mechanism, which describes the data, is a three-step sequential process defined. The sequential character of the mechanism excludes conformational transitions of the DnaB helicase prior to ATP binding. Analysis of relaxation times and amplitudes of the reaction allowed us to estimate all rate and equilibrium constants of partial steps of the proposed mechanism. The intrinsic binding constant for the formation of the (H-ATP) complex is K(ATP) = (1.3 ± 0.5) x 105 M-1. The analysis of the data indicates that a part of the ATP binding energy originates from induced structural changes of the DnaB protein-ATP complex prior to ATP hydrolysis. The equilibrium constant of the chemical interconversion is K(H) = k2/k-2 ~ 2 while the subsequent conformational transition is characterized by K3 = k3/k-3 ~ 30. The low value of K(H) and the presence of the subsequent energetically favorable conformational step(s) strongly suggest that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release. The combined application of single and multiple-turnover approaches show that all six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. Binding of ATP to the DnaB hexamer is characterized by the negative cooperativity parameter σ = 0.25(±0.1). The negative cooperative interactions predominantly affect the ground state of the enzyme-ATP complex. The significance of these results for the mechanism of the free energy transduction of the DnaB helicase is discussed. (C) 2000 Academic Press.",
keywords = "ATPases, DNA replication, Helicases, Protein-nucleotide interactions, Rapid quench-flow kinetics",
author = "Surendran Rajendran and Jezewska, {Maria J.} and Wlodzimierz Bujalowski",
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TY - JOUR

T1 - Multiple-step kinetic mechanism of DNA-independent ATP binding and hydrolysis by Escherichia coli replicative helicase DnaB protein

T2 - Quantitative analysis using the rapid quench-flow method

AU - Rajendran, Surendran

AU - Jezewska, Maria J.

AU - Bujalowski, Wlodzimierz

PY - 2000/11/10

Y1 - 2000/11/10

N2 - The kinetic mechanism of DNA-independent binding and hydrolysis of ATP by the E. coli replicative helicase DnaB protein has been quantitatively examined using the rapid quench-flow technique. Single-turnover studies of ATP hydrolysis, in a non-interacting active site of the helicase, indicate that bimolecular association of ATP with the site is followed by the reversible hydrolysis of nucleotide triphosphate and subsequent conformational transition of the enzyme-product complex. The simplest mechanism, which describes the data, is a three-step sequential process defined. The sequential character of the mechanism excludes conformational transitions of the DnaB helicase prior to ATP binding. Analysis of relaxation times and amplitudes of the reaction allowed us to estimate all rate and equilibrium constants of partial steps of the proposed mechanism. The intrinsic binding constant for the formation of the (H-ATP) complex is K(ATP) = (1.3 ± 0.5) x 105 M-1. The analysis of the data indicates that a part of the ATP binding energy originates from induced structural changes of the DnaB protein-ATP complex prior to ATP hydrolysis. The equilibrium constant of the chemical interconversion is K(H) = k2/k-2 ~ 2 while the subsequent conformational transition is characterized by K3 = k3/k-3 ~ 30. The low value of K(H) and the presence of the subsequent energetically favorable conformational step(s) strongly suggest that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release. The combined application of single and multiple-turnover approaches show that all six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. Binding of ATP to the DnaB hexamer is characterized by the negative cooperativity parameter σ = 0.25(±0.1). The negative cooperative interactions predominantly affect the ground state of the enzyme-ATP complex. The significance of these results for the mechanism of the free energy transduction of the DnaB helicase is discussed. (C) 2000 Academic Press.

AB - The kinetic mechanism of DNA-independent binding and hydrolysis of ATP by the E. coli replicative helicase DnaB protein has been quantitatively examined using the rapid quench-flow technique. Single-turnover studies of ATP hydrolysis, in a non-interacting active site of the helicase, indicate that bimolecular association of ATP with the site is followed by the reversible hydrolysis of nucleotide triphosphate and subsequent conformational transition of the enzyme-product complex. The simplest mechanism, which describes the data, is a three-step sequential process defined. The sequential character of the mechanism excludes conformational transitions of the DnaB helicase prior to ATP binding. Analysis of relaxation times and amplitudes of the reaction allowed us to estimate all rate and equilibrium constants of partial steps of the proposed mechanism. The intrinsic binding constant for the formation of the (H-ATP) complex is K(ATP) = (1.3 ± 0.5) x 105 M-1. The analysis of the data indicates that a part of the ATP binding energy originates from induced structural changes of the DnaB protein-ATP complex prior to ATP hydrolysis. The equilibrium constant of the chemical interconversion is K(H) = k2/k-2 ~ 2 while the subsequent conformational transition is characterized by K3 = k3/k-3 ~ 30. The low value of K(H) and the presence of the subsequent energetically favorable conformational step(s) strongly suggest that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release. The combined application of single and multiple-turnover approaches show that all six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. Binding of ATP to the DnaB hexamer is characterized by the negative cooperativity parameter σ = 0.25(±0.1). The negative cooperative interactions predominantly affect the ground state of the enzyme-ATP complex. The significance of these results for the mechanism of the free energy transduction of the DnaB helicase is discussed. (C) 2000 Academic Press.

KW - ATPases

KW - DNA replication

KW - Helicases

KW - Protein-nucleotide interactions

KW - Rapid quench-flow kinetics

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