Mechanism of Enzymes Involved in Leukotriene C4 Biosynthesis

Cysteinyl leukotrienes (cys-LTs) are potent proinflammatory mediators associated with various diseases including asthma and allergic rhinitis. Leukotriene C4 synthase (LTC4S) and microsomal glutathione transferase 2 (MGST2) catalyze conjugation of the epoxide intermediate LTA4 with GSH to form LTC4, the parent compound of the cys-LTs. Both enzymes belong to the Membrane-Associated Proteins in Eicosanoid and Glutathione metabolism (MAPEG) super family of integral membrane proteins involved in the generation of lipid mediators and in the metabolism of xenobiotics. This thesis investigates the catalytic mechanism and regulation of LTC4S and MGST2. MGST2 can also catalyze conjugation of glutathione (GSH) with electrophilic substrates, such as 1-chloro-2,4-dinitrobenzene (CDNB) and also possesses GSH-dependent peroxidase activity. In this thesis, the overall catalytic mechanism and substrate specificity of human MGST2 has been characterized using purified enzyme. MGST2 (kcat/Km4 = 1.8 × 10 M s) was found to be about 50 times less efficient in catalyzing the biosynthesis of LTC4 compared to LTC4S (kcat/Km4 = 8.7 × 10 M s), while the Km4 for MGST2 (40 μM) and LTC4S (30 μM) were in a similar range. A comparison of LTC4S activity with other GSTs suggests that MGST2 could catalyze conjugation of LTA4 with GSH to form LTC4 under physiological conditions. Both LTC4S and MGST2 bind GSH and activate it to form thiolate anion (GS) at physiological pH and the pKa of enzyme bound GSH was found to be 5.9 and 6.3, respectively. The mechanism of GS formation was characterized for both enzymes using pre-steady-state kinetics. The amplitude analysis of the signals from all different kinetic and spectroscopic experiments suggested that the GS / enzyme subunit stoichiometry was 3/3 for LTC4S and 1/3 for MGST2, which may partly explain the difference in catalytic efficiency. To conclusively show that MGST2 is a functional homo-trimer with one-third-of-the-sites reactivity we combined the results from blue native PAGE, differential scanning calorimetry, isothermal titration calorimetry and equilibrium dialysis followed by global kinetic simulations. Analysis of all microscopic rates and equilibrium constants for GSH binding and activation suggest that GS formation is not a rate-limiting factor for LTC4S, as has been observed for other MAPEG members, such as MGST1. Conversely, GS formation (k2 = 41.1 s) was faster for MGST2 relative to MGST1, but within only one site of the homo-trimer at a given time. Furthermore, pre-steady-state kinetics using CDNB as an electrophilic substrate showed that the chemical conjugation step is most likely rate limiting for MGST2 catalysis under physiological conditions. Recently, a ribosomal S6 kinase (p70S6k) was shown to play a role in the phosphoregulation of LTC4S in monocytes. Here, we identified a major p70S6k phosphorylation site on LTC4S as Ser-36, along with a low-frequency site at Thr-40, by an in vitro phosphorylation assay followed by mass spectrometric analysis. Phosphomimetic mutants were generated to study the functional consequences of phosphorylation by kinetic analysis, molecular dynamics simulations and structural studies. Our results identified Ser-36 as the functionally important site for the regulation of LTC4S activity, where phosphorylation impairs catalytic activity via a mechanism of hydrogen bonding interactions between the phosphoserine and the catalytically important Arg-104, as well as by limiting substrate access to the active site. In summary, MGST2 displays broad substrate specificity similar to MGST1, whereas LTC4S is highly specific towards LTA4 as a physiological substrate. The distinct catalytic and mechanistic properties of MGST2 and LTC4S suggest that while the former may fulfill a promiscuous role in several biochemical pathways, the latter has evolved to fulfill a specific physiological function of LTC4 synthesis. In general, the acquired knowledge about the LTC4S and MGST2 will be useful for the development of pharmaceuticals against inflammatory diseases, and in addition, will provide context during the physiological and mechanistic characterization of other MAPEG members. Moreover, the observed regulation of LTC4S activity through phosphorylation is unique among the MAPEG members and the results presented herein will provide important clues for understanding the mechanism of phosphoregulation during cys-LT biosynthesis. LIST OF SCIENTIFIC PAPERS I. Pre-Steady-State Kinetic Characterization of Thiolate Anion Formation in Human Leukotriene C4 Synthase Agnes Rinaldo-Matthis, Shabbir Ahmad, Anders Wetterholm, Peter Lachmann, Ralf Morgenstern, and Jesper Z. Haeggström. Biochemistry, 2012, 51, 848−856. II. Catalytic Characterization of Human Microsomal Glutathione STransferase 2: Identification of Rate-Limiting Steps Shabbir Ahmad, Damian Niegowski, Anders Wetterholm, Jesper Z. Haeggström, Ralf Morgenstern, and Agnes Rinaldo-Matthis. Biochemistry, 2013, 52, 1755-1764. III. Trimeric microsomal glutathione transferase 2 displays one third of the sites reactivity Shabbir Ahmad, Madhuranayaki Thulasingam, Isolde Palombo, Daniel O. Daley, Kenneth A. Johnson, Ralf Morgenstern, Jesper Z. Haeggström, and Agnes Rinaldo-Matthis. Biochimica et Biophysica Acta, 2015, 1854, 1365-1371. IV. Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity Shabbir Ahmad, A. Jimmy Yetterberg, Madhuranayaki Thulasingam, Fredrik Tholander, Tomas Bergman, Roman Zubarev, Anders Wetterholm, Agnes Rinaldo-Matthis, and Jesper Z. Haeggström. Journal of Biological Chemistry, 2016, 291, 18410-18418. Additional relevant papers o Niegowski, D., Kleinschmidt, T., Ahmad, S., Qureshi, A. A., Marback, M., Rinaldo-Matthis, A., and Haeggstrom, J. Z. (2014) Structure and inhibition of mouse leukotriene C4 synthase. PLoS One , 2014, 9, e96763 o Niegowski, D., Kleinschmidt, T., Olsson, U., Ahmad, S., RinaldoMatthis, A., and Haeggstrom, J. Z. (2014) Crystal structures of leukotriene C4 synthase in complex with product analogs: implications for the enzyme mechanism. J. Biol. Chem. 289, 51995207