Kinetic Parameters Measurement of Human Tyrosylprotein Sulfotransferase using Reverse-Phase Liquid Chromatography coupled to Electrospray Ionization Mass Spectrometry

摘要

Tyrosylprotein sulfotransferase (TPST) catalyzes the transfer of sulfate from the universal sulfate donor adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to the hydroxyl group of a peptidyltyrosine residue to form a tyrosine O-sulfate ester and 3', 5'-ADP (1). Recently, two TPST isoenzymes were identified, TPST 1 and 2 (2,3,4), which are both thought to be responsible for sulfating as much as 1% of the tyrosine residues in the total protein content of a cell (5). Previous studies have determined the kinetic parameters of a mixture of TPSTs using radioactive labeling assays (2,3). In this current research, a novel LC/ESI-MS based TPST2 assay was developed to determine the kinetic constants, Km and Vmax of the reaction.