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3‘-Phosphoadenosine 5‘-Phosphosulfate Binding Site of Flavonol 3-Sulfotransferase Studied by Affinity Chromatography and 31P NMR
- Source :
- Biochemistry. 38:4066-4071
- Publication Year :
- 1999
- Publisher :
- American Chemical Society (ACS), 1999.
-
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.
- 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
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 38
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....bd2260252770fe9f1ce33c40cf7ac582