Enhancement of lipocalin-type prostaglandin D synthase enzyme activity by guanidine hydrochloride.
The characterization of unfolding of mouse recombinant lipocalin-type prostaglandin D synthase (L-PGDS) by guanidine hydrochloride (GdnHCl) was carried out. In the presence of low concentrations of GdnHCl (up to 0.75 M), enhancement of the enzyme activity was observed. However, above a 1 M concentration of GdnHCl, the enzyme activity was reduced in a concentration-dependent manner. The maximum enzyme activity induced by GdnHCl was approximately 1. 5-fold compared with the activity under physiological conditions without GdnHCl. The ellipticity in circular dichroism (CD) spectrum of the L-PGDS at 218 nm, reflecting the beta-sheet content, was decreased by GdnHCl (up to 0.75 M), and the minimum ellipticity was observed at 0.5 M GdnHCl. The fluorescence quenching of the intrinsic tryptophan of L-PGDS due to the binding of bilirubin in the presence or absence of GdnHCl was measured. The K(d) 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 GdnHCl than without it. Further, an NMR study revealed that the reorganization 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 activity of L-PGDS is enhanced by the conformational change, especially by the change in the secondary structure.
Inui
T
T
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Japan.
Ohkubo
T
T
Urade
Y
Y
Hayaishi
O
O
D000818:Animals; D001663:Bilirubin / Q000737:chemistry; D002942:Circular Dichroism; D004789:Enzyme Activation; D005453:Fluorescence; D019791:Guanidine / Q000737:chemistry*; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D054834:Lipocalins; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D054834:Lipocalins; D011994:Recombinant Proteins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D019791:Guanidine; D001663:Bilirubin