PMID:8226921
| Citation |
Furukawa, K, Tagaya, M, Tanizawa, K and Fukui, T (1993) Role of the conserved Lys-X-Gly-Gly sequence at the ADP-glucose-binding site in Escherichia coli glycogen synthase. J. Biol. Chem. 268:23837-42 |
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| Abstract |
Although bacterial and mammalian glycogen synthases differ in the primary structure and specificity for glucosyl donor, lysyl residues identified at their substrate-binding sites by affinity labeling are present in a conserved tetrapeptide sequence, Lys-X-Gly-Gly, where X is a residue not conserved (Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260, 6670-6676; Furukawa, K., Tagaya, M., Inouye, M., Preiss, J., and Fukui, T. (1990) J. Biol. Chem. 265, 2086-2090). To elucidate the functional role of this conserved sequence, Lys-15, Gly-17, and Gly-18 in Escherichia coli glycogen synthase have been replaced by other amino acid residues via site-directed mutagenesis. Kinetic analyses of the Lys-15 mutant enzymes showed that the epsilon-amino group of Lys-15 is mainly involved in binding of the phosphate moiety adjacent to the glycosidic linkage in the substrate ADP-glucose, presumably through an ionic interaction. The mutant enzyme in which Ala was substituted for Gly-17 had a catalytic rate constant 3 orders of magnitude smaller than that of the wild-type enzyme with a slightly increased Michaelis constant for ADP-glucose, whereas the Gly-18-->Ala mutant showed a rate constant only 3.2-fold smaller. In addition, mutations of Gly-17 and Gly-18 resulted in marked changes in the reactivity of Lys-15 with affinity labeling reagents. These results suggest that the 2 glycyl residues in the conserved Lys-X-Gly-Gly sequence, in particular the one closer to the ADP-glucose-binding lysyl residue, participate in catalysis by assisting conformational change(s) of the active site or stabilizing the transition state. |
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| Keywords |
Adenosine Diphosphate Glucose/metabolism; Affinity Labels; Amino Acid Sequence; Base Sequence; Binding Sites; Catalysis; Conserved Sequence; DNA Primers; Escherichia coli/enzymology; Glycine/metabolism; Glycogen Synthase/chemistry; Glycogen Synthase/metabolism; Humans; Kinetics; Lysine/metabolism; Molecular Sequence Data; Mutation |
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