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Journal of Biochemistry Advance Access originally published online on January 20, 2009
Journal of Biochemistry 2009 145(5):585-590; doi:10.1093/jb/mvp012
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Inspection of the Activator Binding Site for 4-{alpha}-Glucanotransferase in Porcine Liver Glycogen Debranching Enzyme with Fluorogenic Dextrins

Eriko Yamamoto, Yumiko Watanabe, Yasushi Makino and Kaoru Omichi*

Department of Chemistry, Graduate School of Science, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan

*To whom correspondence should be addressed. Tel: +81-72-254-9191, E-mail: komichi{at}c.s.osakafu-u.ac.jp

Received November 17, 2008; Accepted January 13, 2009


  Abstract

Recently, we found that {alpha}-, β- and {gamma}-cyclodextrins accelerated the 4-{alpha}-glucanotransferase action of porcine liver glycogen debranching enzyme (GDE) on Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4(Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-6)Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA (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, (Glc{alpha}1-6)mGlc{alpha}1-4(Glc{alpha}1-4)nGlcPA (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 Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4(Glc{alpha}1-6)Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA (B5/81). In the presence of G6PA, the removed maltotriosyl residue was transferred to G6PA to give Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA (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 Glc{alpha}1-6Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc.

Key Words: activation of enzyme, branched dextrin, 4-{alpha}-glucanotransferase, glycogen, glycogen debranching enzyme

Abbreviations: GDE, glycogen debranching enzyme; Glc, D-glucose; GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol; B5/81, Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4(Glc{alpha}1-6)Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA; B5/84, Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4(Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-6)Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA; G8PA, Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA; B7/71, Glc{alpha}1-6Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA; B6/61, Glc{alpha}1-6Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA; B5/51, Glc{alpha}1-6Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA; B5/61, Glc{alpha}1-4(Glc{alpha}1-6)Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA; G9PA, Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4GlcPA; EDTA, ethylenediaminetetraacetate


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