3'-Phosphoadenosine 5'-phosphosulfate binding site of flavonol 3-sulfotransferase studied by affinity chromatography and 31P NMR.
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.
Marsolais
F
F
Department of Biology, Concordia University, Montreal, Quebec, Canada.
Laviolette
M
M
Kakuta
Y
Y
Negishi
M
M
Pedersen
L C
LC
Auger
M
M
Varin
L
L
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008239:Lysine / Q000235:genetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry* / Q000378:metabolism*; D010759:Phosphorus Isotopes; D015238:Sulfotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D010759:Phosphorus Isotopes; D010724:Phosphoadenosine Phosphosulfate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D015238:Sulfotransferases; C072963:flavonol 3-sulfotransferase; D008239:Lysine