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Reaction kinetics of substrate transglycosylation catalyzed by TreX of Sulfolobus solfataricus and effects on glycogen breakdown.

Journal of bacteriology (2014-03-13)
Dang Hai Dang Nguyen, Jong-Tae Park, Jae-Hoon Shim, Phuong Lan Tran, Ershita Fitria Oktavina, Thi Lan Huong Nguyen, Sung-Jae Lee, Cheon-Seok Park, Dan Li, Sung-Hoon Park, David Stapleton, Jin-Sil Lee, Kwan-Hwa Park
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glycogen from Mytilus edulis (Blue mussel), for DNA precipitations
Sigma-Aldrich
Glycogen from oyster, ≥75% dry basis
Sigma-Aldrich
Glycogen from bovine liver, ≥85%
Sigma-Aldrich
Glycogen from rabbit liver, ≥85% dry basis (enzymatic)
Sigma-Aldrich
Glycogen from Mytilus edulis (Blue mussel), ≥85%
Sigma-Aldrich
Glycogen from oyster, Type XI