Ligand Binding and Mechanism of Microsomal Glutathione Transferase 1

The homo-trimeric, membrane bound Microsomal Glutathione Transferase 1 (MGST1, EC. 2.5.1.18) belongs functionally to both the glutathione transferase family (GST) and the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. It is found in high amount in the liver, where it is localised to the endoplasmatic reticulum and the outer membrane of mitochondria. MGST1 possesses both glutathione transferase and peroxidase activity, thus protecting the organism against electrophilic, hydrophobic substances and lipid peroxidation. In this thesis, the rat MGST1 has been used both in purified form from rat livers, heterologously expressed in the bacteria E. coli BL21(DE3) strain and in stably transfected MCF7 cells over expressing the enzyme. MGST1 exhibits one-third-of-the-sites-reactivity towards glutathione, as it is capable of binding three GSH molecules but only stabilising one thiolate anion (GS), the catalytically active form of GSH. Using electrospray mass spectrometry, binding of three GSH molecules was observed within the trimer while the monomer did not bind GSH, in agreement to the proposed binding sites at subunit interfaces (Holm, et al., (2006). J. Mol. Biol. 360(5): 934-945). The binding of GSH could be competed out with equimolar concentration of the inhibitor glutathione sulfonate. Using equilibrium dialysis three bound product molecules (GSDNB) with a global Kd of 320 μM were observed. Using the same technique analysing GSH binding confirmed previous results (Sun et al., (1997). Biochem. J. 326(Pt 1): 193-193) with one GSH bound with a Kd of 16 μM, while competition experiments using GSDNB as a marker for GSH binding showed complete exchange of GSDNB at a few mM GSH. Thus one GSH binds strongly to MGST1 while the other two bind more weakly. To obtain a Kd for more loosely bound GSH, stopped flow experiments were performed and the binding constant for the “third” GSH was determined to 2.5 mM. Hydrogen/deuterium (H/D) exchange has previously been used to determine GSH dependent dynamics in MGST1. GSH dependent changes were found to be localised largely in the cytosol facing regions of the enzyme (Busenlehner et al., (2004). Biochemistry 43(35): 11145-11152). Here, H/D exchange and H/D footprinting was used to further determine the binding sites for GSH and the two putative second substrate binding sites (the hydrophobic, electrophile binding site and the fatty acid/phospholipid binding site, respectively). The two latter sites were found to be localised in different parts of the enzyme and both bordered the GSH binding site. Site directed mutagenesis within the proposed GSH binding site confirmed its location. Other mutational studies revealed that two mutants (R72A and R73Q) lost saturation behaviour for GSH but had extremely high activity at high GSH concentration. These data are also consistent with the proposed GSH binding site location. Finally, a new fluorogenic substrate, based on release of a Rhodamine moiety, has been characterised with purified MGST1 and in MCF7 cells (or cell extracts) expressing MGST1 and was found to be a highly sensitive substrate for MGST1. In conclusion, the studies presented in this thesis yield a deeper understanding of the mechanism and structure of MGST1 as well as providing new experimental tools. LIST OF PUBLICATIONS 1. Richard Svensson, Johan Ålander, Richard N. Armstrong and Ralf Morgenstern. Kinetic characterization of thiolate anion formation and chemical catalysis of activated microsomal glutathione transferase 1. Biochemistry (2004) 43, 8869-8877. 2. Laura S. Busenlehner, Johan Ålander, Caroline Jegerschöld, Peter J. Holm, Priyaranjan Bhakat, Hans Hebert, Ralf Morgenstern and Richard N. Armstrong. Location of Substrate Binding Sites within the Integral Membrane Protein Microsomal Glutathione Transferase-1. Biochemistry (2007) 46, 2812-2822. 3. Johan Ålander, Johan Lengqvist, Peter J. Holm, Richard Svensson, Pascal Gerbaux, Robert H. H. van den Heuvel, Hans Hebert, William J. Griffiths, Richard N. Armstrong and Ralf Morgenstern. Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione. Manuscript. 4. Johan Ålander, Katarina Johansson, Vanina Dahlström Heuser, Henny Farebo, Julia Järvliden Hiroshi Abe, Aya Shibata, Yoshihiro Ito and Ralf Morgenstern. Characterisation of a New Fluorogenic Substrate for Microsomal Glutathione Transferase 1. Submitted. PUBLICATIONS NOT INCLUDED IN THE THESIS 1. Claudia A. Staab, Johan Ålander, Margareta Brandt, Johan Lengqvist, Ralf Morgenstern, Roland C. Grafström and Jan-Olov Höög. Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors. Biochemical Journal (2008) 413, 493-504. 2. Claudia A. Staab, Johan Ålander, Ralf Morgenstern, Roland C. Grafström and Jan-Olov Höög. The Janus face of alcohol dehydrogenase 3. Chemico-Biological Interactions, Special Issue: Enzymology and Molecular Biology of Carbonyl Metabolism, in press.