Human liver thermostable phenol sulfotransferase: photoaffinity labeling with 2-iodo-4-azidophenol.
Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic drugs, neurotransmitters, and xenobiotic compounds. Human tissues contain at least two forms of PST, which differ in their substrate specificities, inhibitor sensitivities, physical properties, and regulation. One form of the enzyme is thermostable (TS) and catalyzes the sulfate conjugation of micromolar concentrations of "simple" phenols. The other form of PST is thermolabile and catalyzes the sulfate conjugation of micromolar concentrations of dopamine and other phenolic monoamines. Quantitative structure-activity relationship (QSAR) analyses of substrate kinetic data obtained with purified human liver TS PST made it possible to design a photoreactive substrate for this form of the enzyme. Because of the very high affinity of TS PST for 2-halogenated phenols, 2-iodo-4-azidophenol (IAP) was synthesized and tested for this purpose. The Km predicted for IAP on the basis of QSAR analysis was 95 nM. The apparent Km determined experimentally was 52 nM. UV irradiation of partially purified human liver TS PST in the presence of [125 I]IAP and 3'-phosphoadenosine-5'-phosphosulfate, the sulfate donor for the reaction, resulted in the radioactive labeling of two proteins, with molecular weights of 32,000 and 34,000, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibitors of the reaction catalyzed by TS PST, including 2,6-dichloro-4-nitrophenol-3'-phosphoadenosine- 5'-phosphate and NaCl, as well as 2-iodophenol, a competing substrate, inhibited the photolabeling of both of these proteins by [125I]IAP in a concentration-dependent fashion. Partially purified TS PST was then radioactively labeled with [125]IAP and was subjected to gel filtration high performance liquid chromatography to verify that the photo-affinity-labeled proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coeluted with TS PST enzyme activity. Photoaffinity labeling of TS PST will be useful in studies of the molecular characteristics of the protein and its active site, as well as in the purification of this important drug-metabolizing enzyme.
Otterness
D M
DM
Department of Pharmacology, Mayo Clinic/Mayo Foundation, Rochester, Minnesota 55905.
Powers
S P
SP
Miller
L J
LJ
Weinshilboum
R M
RM
D000345:Affinity Labels; D015239:Arylsulfotransferase / Q000032:analysis* / Q000302:isolation & purification; D001386:Azides; D002851:Chromatography, High Pressure Liquid; D006358:Hot Temperature; D006801:Humans; D007462:Iodobenzenes / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D009596:Nitrophenols / Q000494:pharmacology; D010782:Photolysis; D014238:Trichloroacetic Acid / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D007462:Iodobenzenes; D009596:Nitrophenols; C062179:2-iodo-4-azidophenol; D014238:Trichloroacetic Acid; C015802:2,6-dichloro-4-nitrophenol; D015239:Arylsulfotransferase; C011289:2-iodophenol