Adenosine 5'-Phosphosulf ate Sulfotransferase from Euglena: Enzyme-Bound Intermediates

Adenosine 5'-phosphosulfate sulfotransferase (APSST) purifiedfrom Euglena gracilis Klebs var. bacillaris mutant W₁₀BSmL byammonium sulfate precipitation, Sephadex G-100 gel filtration,reactive blue agarose, reactive dye agarose and DEAE-cellulosecan be labeled by incubation with AP³⁵S and separated from smallradioactive compounds on Sephadex G-50. Most of the label isnot exchangeable with nonradioactive APS and therefore is notassociated with bound substrate. On non-inactivating SDS-PAGE,a radioactive band at the position of native APSST tetramershows APSST activity (measured as acid-volatile radioactivity).Labeled protein hydrolyzed with Pronase yields radioactive S-sulfocysteine,indicating that at least one cysteine residue of APSST acceptsa sulfo group from APS to form E-S-SO₃^–. A labeled lowmolecular weight compound can be separated from the proteinby paper electrophoresis or by treatment with acidic proteindenaturing reagents such as trifluoroacetic acid (TFA) or trichloroaceticacid (TCA). This labeled compound (perhaps the sulfo-carrier)behaves as a strong acid on paper electrophoresis and is stabilizedby iodoacetamide or acidic conditions but degrades to thiosulfate,sulfate and other compounds as the pH is raised. The radioactivityin APSST is exchangeable with sulfite or thiosulfate. AMP inhibitsAPSST in the formation of acid-volatile radioactivity by competingwith APS, but APA inhibits APSST activity uncompetitively. AK_m of 0.1 µM for APS and K_i of 0.1 mM for AMP and 0.6mM for APA are obtained when a saturating amount of dithiothreitol(DTT) is used as the thiol. A mechanism is proposed for theinitial reaction(s) catalyzed by APSST. ¹Present address: Boyce Thompson Institute for Plant Research,Tower Road, Ithaca, NY 14853, U.S.A.