Sugar binding effects on the enzymatic reaction and conformation near the active site of pokeweed antiviral protein revealed by fluorescence spectroscopy.
In various trials for elucidating the physiological function of pokeweed antiviral protein (PAP), studies on the interaction with sugar are essential. The fluorescence titration curves showed that PAP retained the strong affinity against N-acetylglucosamine (NAG) and two sites in one PAP molecule co-operatively participated in the binding. In the complex of PAP with NAG, Trp208 located at the entrance lid site of substrate came closer to Tyr72 about 0.3 Å. Furthermore, the fluorescence anisotropy decay measurement demonstrated that the segmental rotation of Trp208 was enlarged by the binding of PAP with NAG. Such conformational changes around the active site closely correlate with the enzymatic activity of PAP. The N-glycosidase activity of PAP was enhanced more than two times in the presence of NAG. The obtained results consistently suggested the enzymatic activity of PAP would be regulated through the conformation change near the active site induced by the binding with NAG.
Institute of Biophysics, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan.
Hiromichi
H
Nakashima
Yukihiro
Y
Fukunaga
Ryosuke
R
Ueno
Etsuko
E
Nishimoto
D000117:Acetylglucosamine / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D005454:Fluorescence Polarization; D006026:Glycoside Hydrolases / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D029603:Phytolacca / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D006026:Glycoside Hydrolases; C024275:pokeweed antiviral protein; D000117:Acetylglucosamine