Role of the conserved Lys-X-Gly-Gly sequence at the ADP-glucose-binding site in Escherichia coli glycogen synthase.
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the functional role of this conserved sequence, Lys-15, Gly-17, and Gly-18 in Escherichia coli glycogen synthase have been replaced by other amino acid residues via site-directed mutagenesis.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.
Furukawa
K
K
Institute of Scientific and Industrial Research, Osaka University, Japan.
Tagaya
M
M
Tanizawa
K
K
Fukui
T
T
D000245:Adenosine Diphosphate Glucose / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000378:metabolism; D006006:Glycogen Synthase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation
D000345:Affinity Labels; D017931:DNA Primers; D000245:Adenosine Diphosphate Glucose; D006006:Glycogen Synthase; D008239:Lysine; D005998:Glycine