Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

• Published in: Journal of bacteriology Vol. 196; no. 11; pp. 1941 - 1949
• Main Authors: Nguyen, Dang Hai Dang, Park, Jong-Tae, Shim, Jae-Hoon, Tran, Phuong Lan, Oktavina, Ershita Fitria, Nguyen, Thi Lan Huong, Lee, Sung-Jae, Park, Cheon-Seok, Li, Dan, Park, Sung-Hoon, Stapleton, David, Lee, Jin-Sil, Park, Kwan-Hwa
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.06.2014
• Subjects: Bacterial Proteins - metabolism; Bacteriology; catalytic activity; Chemical reactions; enzyme kinetics; Enzymes; Escherichia coli; Gene Expression Regulation, Bacterial - physiology; Gene Expression Regulation, Enzymologic - physiology; Glucose; glucose 1-phosphate; glycogen; Glycogen - metabolism; Glycogen Debranching Enzyme System - genetics; Glycogen Debranching Enzyme System - metabolism; Glycosylation; Hydrolysis; isomers; liver; phosphorylase; polymerization; rats; Reaction kinetics; Substrate Specificity; Sulfolobus solfataricus; Sulfolobus solfataricus - enzymology; Sulfolobus solfataricus - genetics; Sulfolobus solfataricus - metabolism; yeasts
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/jb.01442-13

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-β-cyclodextrin (Glc4-β-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc4-β-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-β-cyclodextrin [(Glc4)2-β-CD]; these were 61,63- and 61,64-dimaltotetraosyl-α-1,6-β-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the kcat and Km values for transglycosylation were 1.78 × 103 s−1 and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 103 s−1 and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the kcat/Km ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.