Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-sulfotransferase by electrospray ionization FT-ICR mass spectrometry.
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)/Kd(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.
Yu
Yonghao
Y
Department of Chemistry, University of California at Berkeley, 410 Latimer Hall, 94720, Berkeley, CA, USA.
Kirkup
Colleen E
CE
Department of Chemistry, University of California at Berkeley, 410 Latimer Hall, 94720, Berkeley, CA, USA.
Pi
Na
N
Department of Chemistry, University of California at Berkeley, 410 Latimer Hall, 94720, Berkeley, CA, USA.
Leary
Julie A
JA
Department of Chemistry, University of California at Berkeley, 410 Latimer Hall, 94720, Berkeley, CA, USA. leary@socrates.berkeley.edu.
D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D005740:Gases / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D015394:Molecular Structure; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D015238:Sulfotransferases / Q000378:metabolism*; D000097811:Carbohydrate Sulfotransferases
D004187:Disaccharides; D005740:Gases; D008024:Ligands; D011506:Proteins; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C032438:chitobiose