Abstract
Human leukotriene C4 synthase (hLTC4S) is an integral membrane enzyme that conjugates leukotriene (LT) A4 with glutathione to form LTC4, a precursor to the cysteinyl leukotrienes (LTC4, LTD4, and LTE4) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC4S, Arg-104 and Arg-31 have been implicated in the conjugation reaction. Here, we used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3–99.9% of the specific activity against LTA4. Steady-state kinetics of R104A and R104S revealed that the Km for GSH was not significantly affected. UV difference spectra of the binary enzyme-GSH complex indicated that GSH ionization depends on the presence of Arg-104 because no thiolate signal, with λmax at 239 nm, could be detected using R104A or R104S hLTC4S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pKa to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA4. On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC4S by 88 and 70%, respectively, without significantly affecting the kcat/Km values for GSH, and a crystal structure of R31Q hLTC4S (2.1 Å) revealed a Gln-31 side chain pointing away from the active site. We conclude that Arg-104 plays a critical role in the catalytic mechanism of hLTC4S, whereas a functional role of Arg-31 seems more elusive. Because Arg-104 is a conserved residue, our results pertain to other homologous membrane proteins and represent a structure-function paradigm probably common to all microsomal GSH transferases.
Highlights
Human leukotriene C4 synthase2 is a 17-kDa integral membrane-bound enzyme that catalyzes the formation of potent smooth muscle-contracting mediators, the cysteinyl leukotrienes, leukotriene (LT) C4 ((5S)-hydroxy-(6R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid) and its metabolites LTD4 ((5S)-hydroxy-(6R)-S-cysteinylglycyl-7,9trans-11,14-cis-eicosatetraenoic acid) and LTE4 ((5S)-hydroxy-(6R)-S-cysteinyl-7,9-trans-11,14-cis-eicosatetraenoic acid)
This family has six human members and includes three proteins involved in detoxification (MGST1, MGST2, and MGST3) and three proteins with pivotal functions in specific biosynthetic pathways of arachidonic acid metabolism, viz. FLAP, LTC4S, and mPGES-1 (8 –12)
MGST2 and MGST3 are thought to have similar catalytic properties as MGST1, MGST2 has been shown to be able to produce LTC4 (8, 11), but have not yet been assigned any specific function. mPGES-1 catalyzes the oxidoreduction of prostaglandin H2 into prostaglandin E2 and displays low glutathione transferase and glutathione-dependent peroxidase activities (16). Human leukotriene C4 synthase (hLTC4S) is highly substrate-specific compared with the other MAPEG family members or GSH transferases, as it accepts only LTA4, LTA4
Summary
Materials—Imidazole, Tris base, NaCl, KCl, Triton X-100, sodium deoxycholate, S-hexylglutathione-agarose, probenecid, GSH, and 2-mercaptoethanol were obtained from Sigma. The column was washed with 3 column volumes of buffer A (25 mM Tris-HCl (pH 7.8), 10% glycerol, 0.1% Triton X-100, and 5 mM 2-mercaptoethanol) supplemented with 20 mM imidazole and 0.1 M NaCl, followed by an additional wash with buffer A containing 40 mM imidazole and 0.5 M NaCl. hLTC4S was eluted with 300 mM imidazole, 0.5 M NaCl, and 0.1 mM GSH in buffer A. Pure hLTC4S was eluted with 25 mM Tris-HCl (pH 7.8), 0.1% Triton X-100, 30 mM probenecid, 5 mM 2-mercaptoethanol, and 0.1 mM GSH. As a final step for crystallization purposes, the sample was subjected to a buffer exchange on Superdex 200 16/60 (GE Healthcare) equilibrated with 25 mM Tris-HCl (pH 7.2), 0.03% (w/v) dodecyl maltoside, and 300 mM NaCl. Fractions containing R31Q hLTC4S were concentrated to 1–3 mg/ml by ultrafiltration.
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