Adenosine 5′-Phosphosulf ate Sulfotransferase fromEuglena: Enzyme-Bound Intermediates

摘要

Adenosine 5′-phosphosulfate sulfotransferase (APSST) purified fromEuglena gracilisKlebs var.bacillarismutant W10BSmL by ammonium sulfate precipitation, Sephadex G-100 gel filtration, reactive blue agarose, reactive dye agarose and DEAE-cellulose can be labeled by incubation with AP35S and separated from small radioactive compounds on Sephadex G-50. Most of the label is not exchangeable with nonradioactive APS and therefore is not associated with bound substrate. On non-inactivating SDS-PAGE, a radioactive band at the position of native APSST tetramer shows 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 accepts a sulfo group from APS to form E-S-SO3−. A labeled low molecular weight compound can be separated from the protein by paper electrophoresis or by treatment with acidic protein denaturing reagents such as trifluoroacetic acid (TFA) or trichloroacetic acid (TCA). This labeled compound (perhaps the sulfo-carrier) behaves as a strong acid on paper electrophoresis and is stabilized by iodoacetamide or acidic conditions but degrades to thiosulfate, sulfate and other compounds as the pH is raised. The radioactivity in APSST is exchangeable with sulfite or thiosulfate. AMP inhibits APSST in the formation of acid-volatile radioactivity by competing with APS, but APA inhibits APSST activity uncompetitively. AKmof 0.1μM for APS andKiof 0.1 mM for AMP and 0.6 mM for APA are obtained when a saturating amount of dithiothreitol (DTT) is used as the thiol. A mechanism is proposed for the initial reaction(s) catalyzed by A