Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry Na Pi a,b , Mike B. Hoang b , Hong Gao a,b , Joseph D. Mougous c,d , Carolyn R. Bertozzi b,c,d , Julie A. Leary a,b, * a Department of Chemistry and Division of Molecular and Cellular Biology, Genome Center, University of California, Davis, CA 95606, USA b Department of Chemistry, University of California, Berkeley, CA 94720, USA c Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA d Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA Received 12 January 2005 Available online 14 March 2005 Abstract Mycobacterial carbohydrate sulfotransferase Stf0 catalyzes the sulfuryl group transfer from 3 0 -phosphoadenosine-5 0 -phospho- sulfate (PAPS) to trehalose. The sulfation of trehalose is required for the biosynthesis of sulfolipid-1, the most abundant sulfated metabolite found in Mycobacterium tuberculosis. In this paper, an efficient enzyme kinetics assay for Stf0 using electrospray ioniza- tion (ESI) mass spectrometry is presented. The kinetic constants of Stf0 were measured, and the catalytic mechanism of the sulfuryl group transfer reaction was investigated in initial rate kinetics and product inhibition experiments. In addition, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was employed to detect the noncovalent complexes, the Stf0–PAPS and Stf0– trehalose binary complexes, and a Stf0–3 0 -phosphoadenosine 5 0 -phosphate–trehalose ternary complex. The results from our study strongly suggest a rapid equilibrium random sequential Bi-Bi mechanism for Stf0 with formation of a ternary complex intermediate. In this mechanism, PAPS and trehalose bind and their products are released in random fashion. To our knowledge, this is the first detailed mechanistic data reported for Stf0, which further demonstrates the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Stf0; Mass spectrometry; ESI-MS assay; Rapid equilibrium random sequential Bi-Bi mechanism Sulfation of biomolecules is a posttranslational mod- ification that is widely observed from bacteria to mam- mals and regulates a variety of cellular communication events [1,2]. Sulfotransferases (STs) 1 are enzymes that are responsible for transferring the sulfuryl group from a universal sulfuryl group donor, 3 0 -phosphoadenosine 5 0 -phosphosulfate (PAPS), to numerous acceptor sub- strates including proteins [3,4], carbohydrates [1,5], and low-molecular-weight metabolites [6,7]. They also play significant roles in modulating normal and patho- 0003-2697/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.02.004 * Corresponding author. Fax: +1 530 754 9658. E-mail address: jaleary@ucdavis.edu (J.A. Leary). 1 Abbreviations used: ST, sulfotransferase; PAPS, 3 0 -phosphoadenosine 5 0 -phosphosulfate; PAP, 3 0 -phosphoadenosine 5 0 -phosphate; 3 0 PB motif, 3 0 -phosphate binding motif; EST, estrogen sulfotransferase; ER, estrogen receptor; 5 0 PSB-loop, 5 0 -phosphosulfate binding loop; HNDST-1, heparan sulfate N-deacetylase/N-sulfotransferase 1; HS3OST-1, heparan sulfate 3-O-sulfotransferase isoform 1; PST, phenol sulfotransferase; FST, flavonol sulfotransferase; SL-1, sulfolipid-1; T2S, trehalose-2-sulfate; ESI-MS, electrospray ionization mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; FT-ICR MS, Fourier transform ion cyclotron resonance mass spectrometry; GST, glutathione S-transferase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; SIM, selected ion monitoring; GalNAc, N-acetylgalactosamine; DDi-6S, a-DUA-[1 fi 3]- GalNAc-6S. www.elsevier.com/locate/yabio Analytical Biochemistry 341 (2005) 94–104 ANALYTICAL BIOCHEMISTRY