Characterizing human tyrosylprotein sulfotransferases using mass spectrometry

The focus of the research presented in this thesis is on the investigation of human tyrosylprotein sulfotransferases (TPSTs) using a novel mass spectrometry-based TPST assay. Two isozymes of TPST were identified to be present in the trans-Golgi network and were found to be membrane-bound. This has rendered their characterization a challenge compared to the well-studied cytosolic sulfotransferases. Tyrosine sulfation, a protein post-translational modification TPST catalyzes, is known to be essential for efficient protein-ligand binding involved in diverse biological functions. This has made studies on the molecular enzymology of TPSTs of particular interest.;The MS-based TPST assay was used further to investigate the catalytic mechanism of TPST-2 using CCR8 substrates. Through initial rate kinetics, product inhibition studies, and radioactive-labeling, experiments, our data strongly suggest a rapid equilibrium random two-site ping-pong model for TPST-2 catalysis. In this mechanistic model, the enzyme allows independent binding of substrates to two distinct sites and involves the formation of a sulfated enzyme covalent intermediate. Some insights on the important amino acid residues at the catalytic site of TPST-2 and its covalent intermediate are also presented. To our knowledge, this is the first detailed study of the reaction kinetics and mechanism reported for human TPST-2 or any other Golgi-resident sulfotransferase.;In the work presented herein, a quantitative LC/ESI-MS-based TPST assay was developed and applied to study its steady state kinetics. G protein-coupled CC-chemokine receptor 8 (CCR8) peptides that have three tyrosine residues in series were chosen as substrates. This assay is the only method that can directly monitor individual sulfation of tyrosine residues in series and differentiate between mono- and multi-sulfated products, a feature radioactive labeling assays cannot provide. Hence, we were able to compare the kinetic properties of TPST-1, TPST-2, and an equal mixture of TPST-1 and -2 for both mono- and disulfation reactions of the CCR8 substrates. Our results show that the Km,appfor the monosulfated substrate was five-fold less than the nonsulfated substrate for both isozymes and its mixture. The development of this method is the initial step in the investigation of kinetic parameters of the sequential tyrosine sulfation of chemokine receptors by TPSTs.