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Volume 272, Number 21, Issue of May 23, 1997 pp. 13923-13928
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Site-directed Mutagenesis of Human Leukotriene C₄ Synthase

(Received for publication, February 10, 1997, and in revised form, March 20, 1997)

From the Department of Medicine, Harvard Medical School, and the Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, Massachusetts 02115

The functional characteristics of leukotriene C₄ synthase (LTC₄S), which specifically conjugates leukotriene A₄ with GSH, were assessed by mutagenic analysis. Human LTC₄S and the 5-lipoxygenase-activating protein share substantial amino acid identity and predicted secondary structure. The mutation of Arg-51 of LTC₄S to Thr or Ile abolishes the enzyme function, whereas the mutation of Arg-51 to His or Lys provides a fully active recombinant protein. The mutations Y59F, Y97F, Y93F, N55A, V49F, and A52S increase the K_m of the recombinant microsomal enzyme for GSH. The mutation Y93F also markedly reduces enzyme function and increases the optimum for pH-dependent activity. The deletion of the third hydrophobic domain with the carboxyl terminus abolishes the enzyme activity, and function is restored by the substitution of the third hydrophobic domain and carboxyl terminus of 5-lipoxygenase-activating protein for that of LTC₄S. Mutations of C56S and C82V alone or together and the deletion of Lys-2 and Asp-3 of LTC₄S do not alter enzyme function. The direct linkage of two LTC₄S monomers by a 12-amino acid bridge 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 in the catalytic function of LTC₄S, Arg-51 probably opens the epoxide ring and Tyr-93 provides the thiolate anion of GSH. Furthermore, the monomer has independent conjugation activity, and dimerization of LTC₄S maintains the proper protein structure.

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