Two Enzyme Activities of Yeast Glycogen Debranching Enzyme and Their Catalytic Residues.

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Glycogen debranching enzyme (GDE) from Saccharomyces cerevisiae possesses two differentcatalytic activities, 4-α-glucanotransferase/amylo-l, 6-glucosidase, on a single polypeptide chain.To investigate this bifunctional enzyme on the molecule level, GDE gene from S. cerevisiae wascloned and expressed into Escherichia coli, and the best conditions for the enzyme expression wereexamined. When the cultivation temperature of recombinant E. coli strains was lowered to 25°C and the isopropyl-β-D-thiogalactopyranoside (IPTG) concentration used for induction was decreased to as low as 0.02 mM, a total of about 33 mg of recombinant GDE can be isolated from alitre culture. We developed an affinity chromatography using βcyclodextrin immobilizedSepharose-6B (β-CD Sepharose 6B) for purifying GDE. The method requires only a single-steppurification and renders an 87% recovery of the enzyme. Moreover, the purified recombinant GDE is a homogeneous protein and possesses the same characteristics as those of S. cerevisiae. To elucidate the structure-function relationship of yeast GDE, the catalytic residues of the enzyme were determined by sitedirected mutagenesis. Asp535, G1u564 and Asp670 on the Nterminal half, and Asp1086 and Asp1147 on the Cterminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes. Five mutant enzymes, D535 N, E564Q, D670N, D1086N and D1 147N, were constructed. All the purified mutant enzymes possessed either the transferase activity or glucosidase activity. Three mutants, D535N, E564Q and D670N, lost transferase activity but retained glucosidase activity. On the contrary, the other mutants, D1086N and Dl 147N, lost glucosidase activity but retained transferase activity. Furthermore, kinetic parameters of the remaining activity of mutants did not vary markedly from those of wildtype enzyme. These results indicate that the residues, Asp535, GIu564 and Asp670, on the Nterminal half are the catalytic residues of transferase activity and the Asp 1086 and Asp 1147 on the C-terminal half involve glucosidase activity, and provide direct evidence that the transferase and glucosidase of yeast GDE are independent each other.
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Two Enzyme Activities of Yeast Glycogen Debranching Enzyme and Their Catalytic Residues.

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