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

The function of Lys-59, Arg-141, and Arg-277 in PAPS binding and catalysis of the flavonol 3-sulfotransferase was investigated. Affinity chromatograph y of conservative mutants with PAPS analogues allowed us to determine that Lys-59 interacts with the 5' portion of the nucleotide, while Arg-141 inter acts with the 3' portion, confirming assignments deduced from the crystal s tructure of mouse estrogen sulfotransferase [Kakuta, Y., Pedersen, L. G., C arter, 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. P-31 NMR spectra of enzyme-PAP complexes were rec orded for the wild-type enzyme and K59R and K59A mutants. The results of th ese experiments suggest that Lys-59 is involved in the determination of the proper orientation of the phosphosulfate group for catalysis.