STRUCTURAL-SIGNIFICANCE AND FUNCTIONAL-SIGNIFICANCE OF CYSTEINE RESIDUES OF GLUTATHIONE-INDEPENDENT PROSTAGLANDIN-D SYNTHASE - IDENTIFICATION OF CYS(65) AS AN ESSENTIAL THIOL

Glutathione-independent prostaglandin D synthase in rat brain is compo sed of 189 amino acid residues and catalyzes the isomerization of pros taglandin H-2,to prostaglandin D-2, an endogenous sleep-promoting subs tance. This enzyme is the only enzyme among members of the lipocalin s uperfamily composed of various secretory lipophilic ligand-carrier pro teins and is recently identified to be a beta-trace protein, a major c onstituent of human cerebrospinal fluid. We expressed the active enzym e in Escherichia coli and then systematically substituted all cysteine residues of the Delta 1-29 enzyme at positions of 65, 89, and 186 wit h alanine or serine. The parent and mutant enzymes were purified to ap parent homogeneity with a recovery of similar to 30% by chromatography with Sephadex G-50 and S-Sepharose, by which all the enzymes showed i dentical elution profiles. The purified enzymes, irrespective of the m utation, showed almost the same circular dichroism spectral characteri stics as displayed by a highly ordered beta-structure. The recombinant enzymes containing Cys(65) showed the activity comparable with that o f the enzyme purified hom rat brain (similar to 3 mu mol/min/mg of pro tein) in the presence, but not in the absence, of sulfhydryl compounds . However, all of the single, double, and triple mutants without Cys(6 5) lost the enzyme activity. The purified Delta 1-29 Ala(89,186) enzym e was inactivated reversibly by conjugation with glutathione at Cys(65 ) and irreversibly by the stoichiometric chemical modification with N- ethylmaleimide. These results indicate that Cys(65) is an essential th iol of the enzyme and that both the intrinsic and extrinsic sulfhydryl groups are necessary for nonoxidative rearrangement of 9,11-endoperox ide of prostaglandin H-2 to produce prostaglandin D-2 catalyzed by the enzyme.