Pre-steady-state kinetic characterization of thiolate anion formation in human leukotriene C₄ synthase.
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(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 μM). 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.
Department of Medical Biochemistry and Biophysics (MBB), Karolinska Institutet, Stockholm, Sweden.
Agnes
A
Rinaldo-Matthis
Shabbir
S
Ahmad
Anders
A
Wetterholm
Peter
P
Lachmann
Ralf
R
Morgenstern
Jesper Z
JZ
Haeggström
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry; D001667:Binding, Competitive; D055162:Biocatalysis; D020134:Catalytic Domain / Q000187:drug effects; D003545:Cysteine / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D017572:Leukotriene A4 / Q000378:metabolism; D017997:Leukotriene C4 / Q000378:metabolism*; D058990:Molecular Targeted Therapy; D011487:Protein Conformation / Q000187:drug effects; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D017572:Leukotriene A4; D021122:Protein Subunits; D011994:Recombinant Proteins; C469714:glutathionesulfonic acid sodium salt; D017997:Leukotriene C4; D001120:Arginine; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D005978:Glutathione; D003545:Cysteine