Pre-Steady-State Kinetic Characterization of Thiolate Anion Formation in Human Leukotriene C-4 Synthase
2012 (English)In: Biochemistry, ISSN 1520-4995, E-ISSN 0006-2960, Vol. 51, no 4, 848-856 p.Article in journal (Refereed) Published
Human leukotriene C-4 synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C-4 (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 LTA(4), presumably according to an S(N)2 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(-1)). 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) = 14.3 mu M). The release of the thiolate was assessed using a GSH release experiment (1.3 s(-1)). 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(-1)), 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.
Place, publisher, year, edition, pages
2012. Vol. 51, no 4, 848-856 p.
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:su:diva-76337DOI: 10.1021/bi201402sISI: 000300473500003OAI: oai:DiVA.org:su-76337DiVA: diva2:526579