The functional characteristics of leukotriene C-4 synthase (LTC4S), wh
ich specifically conjugates leukotriene A(4) with GSH, were assessed b
y mutagenic analysis. Human LTC4S and the 5-lipoxygenase-activating pr
otein share substantial amino acid identity and predicted secondary st
ructure. The mutation of Arg-51 of LTC4S to Thr or lie abolishes the e
nzyme function, whereas the mutation of Arg-51 to His or Lys provides
a fully active recombinant protein. The mutations Y59F, Y97F, Y93F, N5
5A, V49F, and A52S increase the K-m of the recombinant microsomal enzy
me for GSH. The mutation Y93F also markedly reduces enzyme function an
d increases the optimum for pH-dependent activity. The deletion of the
third hydrophobic domain with the carboxyl terminus abolishes the enz
yme activity, and function is restored by the substitution of the thir
d hydrophobic domain and carboxyl terminus of 5-lipoxygenase-activatin
g protein for that of LTC4S. Mutations of C56S and C82V alone or toget
her and the deletion of Lys-a and Asp-3 of LTC4S do not alter enzyme f
unction. The direct linkage of two LTC4S monomers by a 12-amino acid b
ridge provides an active dimer, and the same bridging of inactive R51I
with a wild-type monomer creates an active pseudo-dimer with function
similar to that of the wild-type enzyme. These results suggest that i
n the catalytic function of LTC4S, Arg-51 probably opens the epoxide r
ing and Tyr-93 provides the thiolate anion of GSH. Furthermore, the mo
nomer has independent conjugation activity, and dimerization of LTC4S
maintains the proper protein structure.