Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry

Mycobacterial carbohydrate sulfo transferase Stf0 catalyzes the sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (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 ionization (ESI) mass spectrometry is presented. The kinetic constants of SO 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'-phosphoadenosine 5'-phosphate-trehalose ternary complex. The results from our study strongly suggest a rapid equilibrium random sequential Bi-Bi mechanism for SO 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 SO, which further demonstrates the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems. (c) 2005 Elsevier Inc. All rights reserved.