Identification of lysine 15 at the active site in Escherichia coli glycogen synthase. Conservation of Lys-X-Gly-Gly sequence in the bacterial and mammalian enzymes.
Glycogen synthases from Escherichia coli and mammalian muscle differ in many respects including regulation, sugar nucleotide specificity, and primary sequence. To compare the structure of the active sites in these enzymes, the affinity-labeling study of the E. coli enzyme was carried out using adenosine diphosphopyridoxal as the reagent. The E. coli enzyme was inactivated in a time- and dose-dependent manner when incubated with the reagent followed by sodium borohydride reduction. The inactivation was markedly protected by ADP-glucose and ADP, suggesting that the reagent was bound to the substrate-binding site. The stoichiometry of the bound reagent to the enzyme was approximately 1:1. Sequence analysis of the labeled peptide isolated from a proteolytic digest of the modified protein revealed that Lys15 is labeled. Based on the geometry of the reagent, the epsilon-amino group of this residue might be located close to the pyrophosphate moiety of ADP-glucose bound to the E. coli enzyme, like that of Lys38 in the rabbit muscle enzyme, which is labeled by uridine diphosphopyridoxal (Tagaya, M., Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 260, 6670-6676; Mahrenholz, A. M., Wang, Y., and Roach, P. J. (1988) J. Biol. Chem. 263, 10561-10567). The importance of the conserved sequence of Lys-X-Gly-Gly is discussed in connection with the glycine-rich region found in many nucleotide-binding proteins.
Furukawa
K
K
Institute of Scientific and Industrial Research, Osaka University, Japan.
Tagaya
M
M
Inouye
M
M
Preiss
J
J
Fukui
T
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006006:Glycogen Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008239:Lysine; D009132:Muscles / Q000201:enzymology; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D006006:Glycogen Synthase; D008239:Lysine