Enhancement of lipocalin-type prostaglandin D synthase enzyme activity by guanidine hydrochloride

The characterization of unfolding of mouse recombinant lipocalin-type prost aglandin D synthase (L-PGDS) by guanidine hydrochloride (GdnHCl) was carrie d out. In the presence of low concentrations of GdnHCl (up to 0.75 M), enha ncement of the enzyme activity was observed. However, above a 1 M concentra tion of GdnHCl, the enzyme activity was reduced in a concentration-dependen t manner. The maximum enzyme activity induced by GdnHCl was approximately 1 .5-fold compared with the activity under physiological conditions without G dnHCl. The ellipticity in circular dichroism (CD) spectrum of the L-PGDS at 218 nm, reflecting the P-sheet content, was decreased by GdnHCl (up to 0.7 5 M), and the minimum ellipticity was observed at 0.5 M GdnHCl. The fluores cence quenching of the intrinsic tryptophan of L-PGDS due to the binding of bilirubin in the presence or absence of GdnHCl was measured. The Kd values obtained in the presence and absence of 0.5 M: GdnHCl were 447 and 115 nM, respectively, indicating lower affinity of the L-PGDS for bilirubin with G dnHCl than without it. Further, an NMR study revealed that the reorganizati on of hydrogen-bond network in the L-PGDS was observed in the presence of 0 .5 M GdnHCl. These results, taken together, indicate that the enzyme activi ty of L-PGDS is enhanced by the conformational change, especially by the ch ange in the secondary structure. (C) 1999 Academic Press.