Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. Stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues

Wlodzimierz Bujalowski, Maria J. Jezewska

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Abstract

The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique. The experiments have been performed with fluorescent ATP and ADP analogues beating the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, εAMP-PNP and εADP. Association of the DnaB helicase with nucleotide cofactors is characterized by four relaxation times that indicate that the binding occurs by a minimum of four- steps. The simplest mechanism which can describe the data is a four-step sequential process where the bimolecular binding step is followed by three isomerization steps. This mechanism is described by the following equation: Helicase + N k1⇆k-1 (H-N)1 k2⇆k-2 (H-N)2 k3⇆k-3 (H-N)3 k4⇆k- 4 (H-N)4. The binding mechanism is independent of the location of the nucleotide cofactor modification and is an intrinsic property of the DnaB helicase-nucleotide system. Quantitative amplitude analyses, using the matrix projection operator technique, allowed us to determine specific fluorescence changes accompanying the formation of all intermediates relative to the fluorescence of the free nucleotide. It shows that the major conformational change of the DnaB helicase-nucleotide complex occurs in the formation of the (H-N)1. Moreover, the value of the bimolecular rate constant, k1, is 3-4 orders of magnitude lower than the value expected for the diffusion- controlled reaction. These results indicate that the determined first step includes formation of the collision and an additional transition of the enzyme-nucleotide complex. The obtained results provide evidence of profoundly different conformational states of the ribose and base regions of the nucleotide-binding site in different intermediates. The sequential nature of the mechanism of the nucleotide binding to the DnaB helicase indicates the lack of the existence of a kinetically significant conformational equilibrium of the helicase protomer and the DnaB hexamer prior to the binding. The significance of these results for the functioning of the DnaB helicase is discussed.

Original languageEnglish (US)
Pages (from-to)2106-2122
Number of pages17
JournalBiochemistry
Volume39
Issue number8
DOIs
StatePublished - Feb 29 2000

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DnaB Helicases
Ribose
Escherichia coli
Nucleotides
Kinetics
Proteins
Fluorescence
Adenosine Diphosphate
Adenylyl Imidodiphosphate
Protein Subunits
Isomerization
Relaxation time
Rate constants
Adenosine Triphosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{57931571ec0e43689457dd6b112fa870,
title = "Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. Stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues",
abstract = "The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique. The experiments have been performed with fluorescent ATP and ADP analogues beating the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, εAMP-PNP and εADP. Association of the DnaB helicase with nucleotide cofactors is characterized by four relaxation times that indicate that the binding occurs by a minimum of four- steps. The simplest mechanism which can describe the data is a four-step sequential process where the bimolecular binding step is followed by three isomerization steps. This mechanism is described by the following equation: Helicase + N k1⇆k-1 (H-N)1 k2⇆k-2 (H-N)2 k3⇆k-3 (H-N)3 k4⇆k- 4 (H-N)4. The binding mechanism is independent of the location of the nucleotide cofactor modification and is an intrinsic property of the DnaB helicase-nucleotide system. Quantitative amplitude analyses, using the matrix projection operator technique, allowed us to determine specific fluorescence changes accompanying the formation of all intermediates relative to the fluorescence of the free nucleotide. It shows that the major conformational change of the DnaB helicase-nucleotide complex occurs in the formation of the (H-N)1. Moreover, the value of the bimolecular rate constant, k1, is 3-4 orders of magnitude lower than the value expected for the diffusion- controlled reaction. These results indicate that the determined first step includes formation of the collision and an additional transition of the enzyme-nucleotide complex. The obtained results provide evidence of profoundly different conformational states of the ribose and base regions of the nucleotide-binding site in different intermediates. The sequential nature of the mechanism of the nucleotide binding to the DnaB helicase indicates the lack of the existence of a kinetically significant conformational equilibrium of the helicase protomer and the DnaB hexamer prior to the binding. The significance of these results for the functioning of the DnaB helicase is discussed.",
author = "Wlodzimierz Bujalowski and Jezewska, {Maria J.}",
year = "2000",
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doi = "10.1021/bi992413m",
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pages = "2106--2122",
journal = "Biochemistry",
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publisher = "American Chemical Society",
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T1 - Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. Stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues

AU - Bujalowski, Wlodzimierz

AU - Jezewska, Maria J.

PY - 2000/2/29

Y1 - 2000/2/29

N2 - The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique. The experiments have been performed with fluorescent ATP and ADP analogues beating the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, εAMP-PNP and εADP. Association of the DnaB helicase with nucleotide cofactors is characterized by four relaxation times that indicate that the binding occurs by a minimum of four- steps. The simplest mechanism which can describe the data is a four-step sequential process where the bimolecular binding step is followed by three isomerization steps. This mechanism is described by the following equation: Helicase + N k1⇆k-1 (H-N)1 k2⇆k-2 (H-N)2 k3⇆k-3 (H-N)3 k4⇆k- 4 (H-N)4. The binding mechanism is independent of the location of the nucleotide cofactor modification and is an intrinsic property of the DnaB helicase-nucleotide system. Quantitative amplitude analyses, using the matrix projection operator technique, allowed us to determine specific fluorescence changes accompanying the formation of all intermediates relative to the fluorescence of the free nucleotide. It shows that the major conformational change of the DnaB helicase-nucleotide complex occurs in the formation of the (H-N)1. Moreover, the value of the bimolecular rate constant, k1, is 3-4 orders of magnitude lower than the value expected for the diffusion- controlled reaction. These results indicate that the determined first step includes formation of the collision and an additional transition of the enzyme-nucleotide complex. The obtained results provide evidence of profoundly different conformational states of the ribose and base regions of the nucleotide-binding site in different intermediates. The sequential nature of the mechanism of the nucleotide binding to the DnaB helicase indicates the lack of the existence of a kinetically significant conformational equilibrium of the helicase protomer and the DnaB hexamer prior to the binding. The significance of these results for the functioning of the DnaB helicase is discussed.

AB - The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique. The experiments have been performed with fluorescent ATP and ADP analogues beating the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, εAMP-PNP and εADP. Association of the DnaB helicase with nucleotide cofactors is characterized by four relaxation times that indicate that the binding occurs by a minimum of four- steps. The simplest mechanism which can describe the data is a four-step sequential process where the bimolecular binding step is followed by three isomerization steps. This mechanism is described by the following equation: Helicase + N k1⇆k-1 (H-N)1 k2⇆k-2 (H-N)2 k3⇆k-3 (H-N)3 k4⇆k- 4 (H-N)4. The binding mechanism is independent of the location of the nucleotide cofactor modification and is an intrinsic property of the DnaB helicase-nucleotide system. Quantitative amplitude analyses, using the matrix projection operator technique, allowed us to determine specific fluorescence changes accompanying the formation of all intermediates relative to the fluorescence of the free nucleotide. It shows that the major conformational change of the DnaB helicase-nucleotide complex occurs in the formation of the (H-N)1. Moreover, the value of the bimolecular rate constant, k1, is 3-4 orders of magnitude lower than the value expected for the diffusion- controlled reaction. These results indicate that the determined first step includes formation of the collision and an additional transition of the enzyme-nucleotide complex. The obtained results provide evidence of profoundly different conformational states of the ribose and base regions of the nucleotide-binding site in different intermediates. The sequential nature of the mechanism of the nucleotide binding to the DnaB helicase indicates the lack of the existence of a kinetically significant conformational equilibrium of the helicase protomer and the DnaB hexamer prior to the binding. The significance of these results for the functioning of the DnaB helicase is discussed.

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