Pre-Steady-State Kinetic Characterization of Thiolate Anion Formation in Human Leukotriene C4 Synthase

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• 作者：Agnes Rinaldo-Matthis ; Shabbir Ahmad ; Anders Wetterholm ; Peter Lachmann ; Ralf Morgenstern ; Jesper Z. Haeggstr枚m
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：January 31, 2012
• 年：2012
• 卷：51
• 期：4
• 页码：848-856
• 全文大小：382K
• 年卷期：v.51,no.4(January 31, 2012)
• ISSN：1520-4995

文摘

Human leukotriene C₄ synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C₄ (LTC₄). This molecule, together with its metabolites LTD₄ and LTE₄, induces inflammatory responses, particularly in asthma, and thus, the enzyme is an attractive drug target. During the catalytic cycle, glutathione (GSH) is activated by hLTC4S that forms a nucleophilic thiolate anion that will attack LTA₄, presumably according to an S_N2 reaction to form LTC₄. We observed that GSH thiolate anion formation is rapid and occurs at all three monomers of the homotrimer and is concomitant with stoichiometric release of protons to the medium. The pK_a (5.9) for enzyme-bound GSH thiol and the rate of thiolate formation were determined (k_obs = 200 s^鈥?). Taking advantage of a strong competitive inhibitor, glutathionesulfonic acid, shown here by crystallography to bind in the same location as GSH, we determined the overall dissociation constant (K_d^{GS鈥?/sup> = 14.3 渭M). The release of the thiolate was assessed using a GSH release experiment (1.3 s鈥?). Taken together, these data establish that thiolate anion formation in hLTC4S is not the rate-limiting step for the overall reaction of LTC₄ production (k_cat = 26 s鈥?), and compared to the related microsomal glutathione transferase 1, which displays very slow GSH thiolate anion formation and one-third of the sites reactivity, hLTC4S has evolved a different catalytic mechanism.}