The Catalytic Architecture of Leukotriene C4 Synthase with Two Arginine Residues

Abstract

Leukotriene (LT) C4 and its metabolites, LTD4 and LTE4, are involved in the pathobiology of bronchial asthma. LTC4 synthase is the nuclear membrane-embedded enzyme responsible for LTC4 biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA4 as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA4 binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA4.

A leukotriene C4 synthase inhibitor with the backbone of 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid

“…The estimated K m for GSH and LTA 4 -Me were 0.27 AE 0.05 mM and 5.0 AE 0.3 mM, respectively. These K m values are comparable with those in our previous report (19).…”

“…This reversible suppression shows that the compounds do not impose denaturation or aggregation of the homotrimer of LTC 4 S. However, there may be some structural changes that are implied by the compounddependent Hill slope variation. The crystal structure of the homo-trimer of LTC 4 S bound GSH showed that LTC 4 S forms three equivalent active sites between interfaces of homo-trimer (17,19). The binding of GSH as the natural substrate stabilizes the homo-trimer of LTC 4 S (25).…”

“…The proteins for the enzyme assay were prepared in the same method as described in our previous report on the enzyme mechanism of LTC 4 S including kinetic analysis and high resolution crystal structure analysis (19). LTC 4 S from a Superose-12 column equilibrated with a solution of 20 mM MES-NaOH (pH 6.5), 0.1 M NaCl, 0.04% (w/v) dodecyl-b-D-maltoside (DDM), 1 mM DTT, 10% (v/v) glycerol and 5 mM GSH, was concentrated to $5 mg/ml, then stored at À80 C. Concentrations of the purified enzymes were determined based on UV absorption at 280 nm, and the milligram extinction coefficient 1.57 mg À1 cm À1 was used.…”

“…The quantification of LTC 4 -Me was performed using RP-HPLC with a SYSTEM GOLD 126 solvent module, a 168 detector, a 508 autosampler (Beckman-Coulter) and a YMC-Pack PolymerC18 (4.6 mm  250 mm, S-6 mm) (19). The column was equilibrated with solvent A at a flow rate of 1 ml/min.…”

Enzymes of the Cyclooxygenase Pathways of Prostanoid Biosynthesis

“…The important roles of Arg-31 and Arg-104 were recently experimentally verified. 315,316 In marked contrast, the cleft between TM1 and TM4 of adjacent monomers was not recognizable in the structure of mPGES-1. 309 Thus, the most likely entry point for PGH 2 reaching the glutathione was blocked and no alternative paths were found.…”