Observation of a hybrid random ping‐pong mechanism of catalysis for NodST: A mass spectrometry approach

Abstract

An efficient enzyme kinetics assay using electrospray ionization mass spectrometry (ESI-MS) was initially applied to the catalytic mechanism investigation of a carbohydrate sulfotransferase, NodST. Herein, the recombinant NodST was overexpressed with a His 6 -tag and purified via Ni-NTA metal-affinity chromatography. In this bisubstrate enzymatic system, an internal standard similar in structure and ionization efficiency to the product was chosen in the ESI-MS assay, and a single point normalization factor was determined and used to quantify the product concentration. The catalytic mechanism of NodST was rapidly determined by fitting the MS kinetic data into a nonlinear regression analysis program. The initial rate kinetics analysis and product inhibition study described support a hybrid double-displacement, two-site ping-pong mechanism of NodST with formation of a sulfated NodST intermediate. This covalent intermediate was further isolated and detected via trypsin digestion and Fourier transform ion cyclotron resonance mass spectrometry. To our knowledge, these are the first mechanistic data reported for the bacterial sulfotransferase, NodST, which demonstrated the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems. Keywords:NodST; mass spectrometry; ESI-MS assay; hybrid random ping-pong mechanism Sulfotransferases are enzymes that install the sulfuryl group from 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS), a ubiquitous sulfate group donor, to numerous acceptor substrates including proteins (Farzan et al. 1999;Kehoe and Bertozzi 2000), carbohydrates (Bowman and Bertozzi 1999;Habuchi 2000), and small molecules. This particular reaction, referred to as sulfuryl group transfer, is widely observed from bacteria to mammals and plays a key role in a variety of cellular communication events (Bowman and Bertozzi 1999;Armstrong and Bertozzi 2000). For example, the tyrosine sulfation in chemokine receptor CCR5 is important in the HIV infection process, and the sulfation of estradiol, catalyzed by estrogen sulfotransferase (EST), can activate the estrogen receptor (ER) and is believed to underlie the genesis of breast cancer. The interaction between L-selectin and its sulfated glycoprotein ligands contributes to the leukocyte immigration into inflamed tissues and results in acute and chronic inflammation (Bistrup et al. 1999;Hiraoka et al. 1999). Thus, sulfotransferases play a significant role in modulating normal and pathogenic biological processes and are considered to be a novel class of therapeutic targets (Bowman and Bertozzi 1999;Armstrong and Bertozzi 2000).The catalytic mechanism of sulfuryl group transfer has recently become the focus of intense interest due to its importance in understanding the role of sulfotransferases in vivo. Crystallographic and mutational analyses have provided critical information about the active-site residues inReprint requests to: Julie A. Leary, Department of Chemistry, University of California, Berkeley, CA 9...

Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-sulfotransferase by electrospray ionization FT-ICR mass spectrometry

“…In so doing, we have successfully identified a sulfated enzyme intermediate, which was predicted by our previous study of NodST catalytic mechanism using an ESI-MS kinetic assay [23]. It is also shown that information regarding solution binding affinities can be obtained in the gas phase using ESI-FTICR mass spectrometry.…”

“…In a similar manner, NodST can utilize chitobiose as the acceptor substrate to produce sulfated chitobiose (Scheme 1). Previous studies have shown that the substrate, PAPS, and the product inhibitor, 3Ј-phosphoadenosine 5Ј-phosphate (PAP), bind to NodST with the binding constants of 6.7 M and 1.8 M, respectively [22,23]. Additionally, mass spectrometry was used to show that NodST catalyzes the sulfation using a random hybrid ping-pong Bi-Bi mechanism, supporting the formation of a sulfated-NodST intermediate [23].…”

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

“…Esaki et al reported a novel catalytic mechanism for L-2-Haloacid dehalogenase involving a cyanoalanine intermediate revealed by LC/MS monitoring of the enzymatic reaction [16]. Likewise, Leary and coworkers reported the identification of a sulfated NodH sulfotransferase (NodST) intermediate formed in a hybrid Ping-Pong mechanism using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry [17]. A facile and broadly applicable ESI-MS assay developed by Leary and coworkers has been used for kinetic analyses on several enzyme systems such as glutathione S-transferase (GST) [27], hexokinase [28], and NodH sulfotransferase (NodST) [29].…”

“…The mass spectrometry assay is especially useful because traditional spectrophotometric assays are not applicable to NodST kinetics since no shift in absorption accompanies sulfuryl group transfer. Using this assay, precise and accurate kinetic data were generated for all the enzymes above, and for the first time, a hybrid Ping-Pong Bi-Bi mechanism was unraveled for NodST [17]. A distinct feature of the ESI-MS assay is that product quantification is realized by monitoring the product ion in relation to an internal standard ion in a quenched reaction solution, allowing different product ions of different mass to charge ratio to be monitored and quantified simultaneously in one reaction system.…”

Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton

“…Recently, the development of soft ionization techniques, such as electrospray ionization (ESI), has made MS an excellent complementary technique to conventional methods for studying enzyme kinetics (24)(25)(26)(27)(28)(29)(30). A facile and broadly applicable ESI͞MS assay developed by Leary and coworkers has been implemented for kinetic analyses and substrate specificity evaluation of several enzyme systems such as glutathione S-transferase (26), hexokinase (27), phosphoglucomutase (28), and NodH sulfotransferase (29,30). Herein, a somewhat different ESI͞MS technique was used to develop assays for NovL and NovM individually and in tandem.…”