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Journal of Biochemistry Advance Access originally published online on February 21, 2007
Journal of Biochemistry 2007 141(5):627-634; doi:10.1093/jb/mvm065
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© 2007 The Japanese Biochemical Society.

Active Site Mapping of Amylo-{alpha}-1,6-glucosidase in Porcine Liver Glycogen Debranching Enzyme Using Fluorogenic 6-O-{alpha}-Glucosyl-maltooligosaccharides

Eriko Yamamoto, Yasushi Makino and Kaoru Omichi*

Department of Chemistry, Graduate School of Science, Osaka Prefecture University, 2-1, Daisen-cho, Sakai, Osaka 590-0035, Japan

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

Received December 24, 2006; Accepted February 13, 2007


  Abstract

Glycogen debranching enzyme (GDE) has two enzymatic activities, 4-{alpha}-glucanotransferase and amylo-{alpha}-1,6-glucosidase. Products with 6-O-{alpha}-glucosyl structures formed from phosphorylase limit dextrin by the 4-{alpha}-glucanotransferase activity are hydrolyzed to glucose by the amylo-{alpha}-1,6-glucosidase activity. Here, we probed the active site of amylo-{alpha}-1,6-glucosidase in porcine liver GDE using various 6-O-{alpha}-glucosyl-pyridylamino (PA)-maltooligosaccharides, with structures (Glc{alpha}1-4)m(Glc{alpha}1-6)Glc{alpha}1-4(Glc{alpha}1-4)nGlcPA (GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol residue). Fluorogenic dextrins were prepared from 6-O-{alpha}-glucosyl-{alpha}-, ß-, or {gamma}-cyclodextrin through partial acid hydrolysis, followed by fluorescent tagging of the reducing-end residues of the hydrolysates and separation by gel filtration and reversed-phase HPLC. Porcine liver GDE hydrolyzed dextrins with the structure Glc{alpha}1-4(Glc{alpha}1-6)Glc{alpha}1-4Glc to glucose and the corresponding PA-maltooligosaccharides, whereas other dextrins were not hydrolyzed. Thus, substrates must have two glucosyl residues sandwiching the isomaltosyl moiety to be hydrolyzed. The rate of hydrolysis increased as m increased and reached maximum at m = 4. The rates were the highest when n = 1 but did not vary much with changes in n. Of the dextrins examined, Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4Glc{alpha}1-4(Glc{alpha}1-6)Glc{alpha}1-4Glc{alpha}1-4GlcPA (63-O-{alpha}-glucosyl-PA-maltoheptaose) was hydrolyzed most rapidly, suggesting that it fits the best in the amylo-{alpha}-1,6-glucosidase active site. It is likely that the active site accommodates 62-O-{alpha}-glucosyl-maltohexaose and that the interactions of seven glucosyl residues with the active site allow the most rapid hydrolysis of the {alpha}-1,6-glucosidic linkage of the isomaltosyl moiety.

Key Words: active site, amylo-{alpha}-1,6-glucosidase, branched maltooligosaccharide, fluorogenic substrate, glycogen debranching enzyme

Abbreviations: FD, fluorogenic dextrin; GDE, glycogen debranching enzyme; GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol residue; PA, pyridylamino; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry


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Y. Watanabe, Y. Makino, and K. Omichi
Donor Substrate Specificity of 4-{alpha}-Glucanotransferase of Porcine Liver Glycogen Debranching Enzyme and Complementary Action to Glycogen Phosphorylase on Debranching
J. Biochem., March 1, 2008; 143(3): 435 - 440.
[Abstract] [Full Text] [PDF]


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