3'-Phosphoadenosine 5'-phosphosulfate binding site of flavonol 3- sulfotransferase studied by affinity chromatography and 31P NMR

Frédéric Marsolais, Mario Laviolette, Yoshimitsu Kakuta, Masahiko Negishi, Lars C. Pedersen, Michèle Auger, Luc Varin

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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.

Original languageEnglish
Pages (from-to)4066-4071
Number of pages6
JournalBiochemistry
Volume38
Issue number13
DOIs
Publication statusPublished - Mar 30 1999
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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