Association of bi-functional activity in the N-terminal domain of glycogen debranching enzyme

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• 作者：Lee Min-ho^a ; ^b ; Song Hyung-Nam^b ; Cho Ji-Eun^b ; Tran Phuong Lan^c ; Park Sunghoon^d ; Park Jong-Tae^e ; Woo Eui-Jeon^a ; ^b ; ^{ejwoo@kribb.re.kr" class="auth_mail}
• 关键词：Saccharomyces cerevisiae ; Glycogen debranching enzyme (GDE) ; 伪-1 ; 4-Transferase activity ; 伪-1 ; 6-Glucosidase activity ; Bi-functional
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：28 February, 2014
• 年：2014
• 卷：445
• 期：1
• 页码：107-112
• 全文大小：1010 K

文摘

Glycogen debranching enzyme (GDE) in mammals and yeast exhibits 伪-1,4-transferase and 伪-1,6-glucosidase activities within a single polypeptide chain and facilitates the breakdown of glycogen by a bi-functional mechanism. Each enzymatic activity of GDE is suggested to be associated with distinct domains; 伪-1,4-glycosyltransferase activity with the N-terminal domain and 伪-1,6-glucosidase activity with the C-terminal domain. Here, we present the biochemical features of the GDE from Saccharomyces cerevisiae using the substrate glucose(n)-尾-cyclodextrin (Gn-尾-CD). The bacterially expressed and purified GDE N-terminal domain (aa 1-644) showed 伪-1,4-transferase activity on maltotetraose (G4) and G4-尾-CD, yielding various lengths of (G)_n. Surprisingly, the N-terminal domain also exhibited 伪-1,6-glucosidase activity against G1-尾-CD and G4-尾-CD, producing G1 and 尾-CD. Mutational analysis showed that residues D535 and E564 in the N-terminal domain are essential for the transferase activity but not for the glucosidase activity. These results indicate that the N-terminal domain (1-644) alone has both 伪-1,4-transferase and the 伪-1,6-glucosidase activities and suggest that the bi-functional activity in the N-domain may occur via one active site, as observed in some archaeal debranching enzymes.