SITE-DIRECTED MUTAGENESIS OF HUMAN LEUKOTRIENE C-4 SYNTHASE

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.