title: Reaction kinetics of substrate transglycosylation catalyzed by TreX of Sulfolobus solfataricus and effects on glycogen breakdown.
abstract: 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 6(1),6(3)- and 6(1),6(4)-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 × 10(3) s(-1) and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10(3) 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.
authors.affiliation: Department of Foodservice Management and Nutrition, Sangmyung University, Seoul, Republic of Korea.
authors.forename: Dang Hai Dang
authors.initials: DH
authors.lastname: Nguyen
authors.forename: Jong-Tae
authors.initials: JT
authors.lastname: Park
authors.forename: Jae-Hoon
authors.initials: JH
authors.lastname: Shim
authors.forename: Phuong Lan
authors.initials: PL
authors.lastname: Tran
authors.forename: Ershita Fitria
authors.initials: EF
authors.lastname: Oktavina
authors.forename: Thi Lan Huong
authors.initials: TL
authors.lastname: Nguyen
authors.forename: Sung-Jae
authors.initials: SJ
authors.lastname: Lee
authors.forename: Cheon-Seok
authors.initials: CS
authors.lastname: Park
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Sung-Hoon
authors.initials: SH
authors.lastname: Park
authors.forename: David
authors.initials: D
authors.lastname: Stapleton
authors.forename: Jin-Sil
authors.initials: JS
authors.lastname: Lee
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006003:Glycogen / Q000378:metabolism*; D006004:Glycogen Debranching Enzyme System / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D006004:Glycogen Debranching Enzyme System; D006003:Glycogen