Catalytic mechanism of Golgi-resident human tyrosylprotein sulfotransferase-2: A mass spectrometry approach

Abstract

Human tyrosylprotein sulfotransferases catalyze the transfer of a sulfuryl moiety from the universal sulfate donor PAPS to the hydroxyl substituent of tyrosine residues in proteins and peptides to yield tyrosine sulfated products and PAP. Tyrosine sulfation occurs in the trans-Golgi network affecting an estimated 1% of the tyrosine residues in all secreted and membrane-bound proteins in higher order eukaryotes. In this paper, an effective LC-MS-based TPST kinetics assay was developed and utilized to measure the kinetic properties of human TPST-2 and investigate its catalytic mechanism when G protein-coupled CC-chemokine receptor 8 (CCR8) peptides were used as acceptor substrates. Through initial rate kinetics, product inhibition studies, and radioactive-labeling experiments, our data strongly suggest a two-site ping-pong model for TPST-2 action. 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.

Structural plasticity in the human cytosolic sulfotransferase dimer and its role in substrate selectivity and catalysis

“…After the first product is released, the active form of the enzyme is then free to bind the second substrate, catalyze the reaction, and release the final product. The membrane-bound sulfotransferase superfamily (STs) acts through this ping-pong mechanism [76]. In contrast, aside from a few early studies, kinetic studies have defined the SULTs as proceeding via a sequential mechanism where a ternary complex forms before catalysis and product release [77].…”

Preparation and Analysis of N-Terminal Chemokine Receptor Sulfopeptides Using Tyrosylprotein Sulfotransferase Enzymes

“…A major impurity in any PAPS preparation is 3′-phosphoadenosine-5′-phosphate (PAP), which is formed by spontaneous hydrolysis of PAPS. As a by-product of the TPST-catalyzed sulfotransfer reaction PAP acts as a product inhibitor of tyrosine sulfation (Danan, Yu, Hoffhines, Moore et al, 2008; Danan, Yu, Ludden, Jia et al, 2010), which is one reason why sulfation rates of in vitro sulfation reactions decrease over time. Thus, for efficient in vitro sulfation of peptide substrates, in particular, if multiple sulfation sites are present, high-purity (≥80%) PAPS preparations with low (<10%) PAP content are to be used.…”

“…An added benefit of using TPSTs to produce sulfopeptides is that the reactions can be monitored overtime and, if multiple tyrosines are present, the order of tyrosine sulfation can be determined. This can be done simply by quantifying absorbance of RP-HPLC peaks (Seibert et al, 2002; Seibert, Veldkamp, et al, 2008) or through monitoring the reactions using more complex mass spectrometry approaches pioneered by Leary and colleagues (Danan et al, 2008; Danan et al, 2010; Jen et al, 2009). Knowledge of tyrosine sulfation order provides information of which sulfopeptides are most physiologically relevant for study and such kinetic data can also provide information on the enzymatic mechanism of TPSTs (Danan et al, 2008; Danan et al, 2010; Jen et al, 2009).…”

Fragment-Based Optimization of Small Molecule CXCL12 Inhibitors for Antagonizing the CXCL12/CXCR4 Interaction

“…Using NMR to monitor the titration of sulfotyrosine into four representative chemokines, we observed a sulfotyrosine recognition site analogous to the cleft on CXCL12 that binds sY21 of the receptor CXCR4 [65]. With an estimated 1% of all tyrosines in cell-surface and secreted human proteins modified by O -sulfation [66], inhibitor development against sY binding sites can have far reaching implications in targeting disease-related protein-protein interfaces. The discovery and optimization of a “privileged” small-molecule scaffold would be invaluable for the development of ligands against other sulfotyrosine recognition sites.…”