Site-directed mutagenesis of human leukotriene C4 synthase.
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 Km 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.
Lam
B K
BK
Department of Medicine, Harvard Medical School, and the Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Penrose
J F
JF
Xu
K
K
Baldasaro
M H
MH
Austen
K F
KF
D058927:5-Lipoxygenase-Activating Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017384:Sequence Deletion
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; D001216:Asparagine; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D005978:Glutathione; D003545:Cysteine