101. 3‘-Phosphoadenosine 5‘-Phosphosulfate Binding Site of Flavonol 3-Sulfotransferase Studied by Affinity Chromatography and 31P NMR
- Author
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Frédéric Marsolais, Michèle Auger, Yoshimitsu Kakuta, Masahiko Negishi, Luc Varin, Mario Laviolette, and Lars C. Pedersen
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Macromolecular Substances ,Stereochemistry ,Phosphoadenosine Phosphosulfate ,Mutant ,Biochemistry ,Chromatography, Affinity ,Mice ,Adenosine Triphosphate ,Affinity chromatography ,Animals ,Nucleotide ,Estrogen Sulfotransferase ,Enzyme Inhibitors ,Conserved Sequence ,3'-phosphoadenosine 5'-phosphosulfate binding ,chemistry.chemical_classification ,Binding Sites ,Chemistry ,Negishi coupling ,Lysine ,Phosphorus Isotopes ,Adenosine Diphosphate ,Enzyme ,Sulfotransferases ,Nucleoside - Abstract
The function of Lys-59, Arg-141, and Arg-277 in PAPS binding and catalysis of the flavonol 3-sulfotransferase was investigated. Affinity chromatography of conservative mutants with PAPS analogues allowed us to determine that Lys-59 interacts with the 5' portion of the nucleotide, while Arg-141 interacts with the 3' portion, confirming assignments deduced from the crystal structure of mouse estrogen sulfotransferase [Kakuta, Y., Pedersen, L. G., Carter, C. W. , Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904-908]. The affinity chromatography method could be used to characterize site-directed mutants for other types of enzymes that bind nucleoside 3',5'- or 2',5'-diphosphates. 31P NMR spectra of enzyme-PAP complexes were recorded for the wild-type enzyme and K59R and K59A mutants. The results of these experiments suggest that Lys-59 is involved in the determination of the proper orientation of the phosphosulfate group for catalysis.
- Published
- 1999