Inspection of the activator binding site for 4-alpha-glucanotransferase in porcine liver glycogen debranching enzyme with fluorogenic dextrins.
Recently, we found that alpha-, beta- and gamma-cyclodextrins accelerated the 4-alpha-glucanotransferase action of porcine liver glycogen debranching enzyme (GDE) on Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/84), and proposed the presence of an activator binding site in the GDE molecule. In liver cells, the structures of alpha-glucans proximal to the site GDE acts are not cyclodextrins, but glycogen and its degradation products. To estimate the structural characteristics of intrinsic activators and to inspect the features of the activator binding site, we examined the effects of four fluorogenic dextrins, (Glcalpha1-6)(m)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (B5/51, m = 1, n = 3; B6/61, m = 1, n = 4; B7/71, m = 1, n = 5; G6PA, m = 0, n = 4), on the debranching of B5/84 by porcine liver GDE. The GDE 4-alpha-glucanotransferase removed the maltotriosyl residue from the maltotetraosyl branch of B5/84, producing Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/81). In the presence of G6PA, the removed maltotriosyl residue was transferred to G6PA to give Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (G9PA). In the absence of G6PA, the removed maltotriosyl residue was transferred to water. B7/71, B6/61 and B5/51 did not undergo any changes by the GDE, but they accelerated the action of the 4-alpha-glucanotransferase in removing the maltotriosyl residue. Of the four fluorogenic dextrins examined, B6/61 most strongly accelerated the 4-alpha-glucanotransferase action. The activator binding site is likely to be a space that accommodates the structure of Glcalpha1-6Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glc.
Yamamoto
Eriko
E
Department of Chemistry, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka, Japan.
Watanabe
Yumiko
Y
Makino
Yasushi
Y
Omichi
Kaoru
K
D000818:Animals; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D003912:Dextrins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006004:Glycogen Debranching Enzyme System / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D034421:Sus scrofa
D003912:Dextrins; D005456:Fluorescent Dyes; D006004:Glycogen Debranching Enzyme System; D011134:Polysaccharides; C020569:4 alpha-glucanotransferase