Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-Sulfotransferase by electrospray ionization FT-ICR mass spectrometry

Yonghao Yu, Colleen E. Kirkup, Na Pi, Julie A. Leary

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33 Citations (Scopus)

Abstract

In this study, a GlcNAc-6-O-Sulfotransferase, NodST and its complexation with the substrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and the inhibitor 3′-phosphoadenosine 5′-phosphate (PAP) were studied using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. In addition, using isotopically labeled substrate, we have successfully confirmed a sulfated enzyme intermediate, which was predicted by the MS kinetic measurement. It is also shown that information regarding solution binding affinities can be obtained using electrospray ionization (ESI)-FTICR mass spectrometry. The relative binding constants, Kd(PAPS)/K d(PAP), derived from the solution and gas phase were very similar, which suggests that the binding domain of this particular enzyme system, given known structures of other sulfotransferases, may be preserved during the transmission of the complex from solution to the gas phase.

Original languageEnglish (US)
Pages (from-to)1400-1407
Number of pages8
JournalJournal of the American Society for Mass Spectrometry
Volume15
Issue number10
DOIs
StatePublished - Oct 2004

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Electrospray ionization
Phosphoadenosine Phosphosulfate
Mass spectrometry
Cyclotrons
Cyclotron resonance
Mass Spectrometry
Fourier Analysis
Ligands
Fourier transforms
Enzymes
Gases
Ions
Sulfotransferases
Proteins
Substrates
Complexation
Kinetics
carbohydrate sulfotransferases
adenosine 3'-phosphate-5'-phosphate

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

Cite this

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title = "Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-Sulfotransferase by electrospray ionization FT-ICR mass spectrometry",
abstract = "In this study, a GlcNAc-6-O-Sulfotransferase, NodST and its complexation with the substrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and the inhibitor 3′-phosphoadenosine 5′-phosphate (PAP) were studied using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. In addition, using isotopically labeled substrate, we have successfully confirmed a sulfated enzyme intermediate, which was predicted by the MS kinetic measurement. It is also shown that information regarding solution binding affinities can be obtained using electrospray ionization (ESI)-FTICR mass spectrometry. The relative binding constants, Kd(PAPS)/K d(PAP), derived from the solution and gas phase were very similar, which suggests that the binding domain of this particular enzyme system, given known structures of other sulfotransferases, may be preserved during the transmission of the complex from solution to the gas phase.",
author = "Yonghao Yu and Kirkup, {Colleen E.} and Na Pi and Leary, {Julie A.}",
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T1 - Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-Sulfotransferase by electrospray ionization FT-ICR mass spectrometry

AU - Yu, Yonghao

AU - Kirkup, Colleen E.

AU - Pi, Na

AU - Leary, Julie A.

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N2 - In this study, a GlcNAc-6-O-Sulfotransferase, NodST and its complexation with the substrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and the inhibitor 3′-phosphoadenosine 5′-phosphate (PAP) were studied using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. In addition, using isotopically labeled substrate, we have successfully confirmed a sulfated enzyme intermediate, which was predicted by the MS kinetic measurement. It is also shown that information regarding solution binding affinities can be obtained using electrospray ionization (ESI)-FTICR mass spectrometry. The relative binding constants, Kd(PAPS)/K d(PAP), derived from the solution and gas phase were very similar, which suggests that the binding domain of this particular enzyme system, given known structures of other sulfotransferases, may be preserved during the transmission of the complex from solution to the gas phase.

AB - In this study, a GlcNAc-6-O-Sulfotransferase, NodST and its complexation with the substrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and the inhibitor 3′-phosphoadenosine 5′-phosphate (PAP) were studied using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. In addition, using isotopically labeled substrate, we have successfully confirmed a sulfated enzyme intermediate, which was predicted by the MS kinetic measurement. It is also shown that information regarding solution binding affinities can be obtained using electrospray ionization (ESI)-FTICR mass spectrometry. The relative binding constants, Kd(PAPS)/K d(PAP), derived from the solution and gas phase were very similar, which suggests that the binding domain of this particular enzyme system, given known structures of other sulfotransferases, may be preserved during the transmission of the complex from solution to the gas phase.

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