title: Identification of Lys277 at the active site of Escherichia coli glycogen synthase. Application of affinity labeling combined with site-directed mutagenesis.
abstract: Lys15 in Escherichia coli glycogen synthase, which is specifically labeled by adenosine diphosphopyridoxal, is mainly involved in binding of the substrate ADP-glucose (Furukawa, K., Tagaya, M., Tanizawa, K., and Fukui, T. (1993) J. Biol. Chem. 268, 23837-23842). We have found that the mutant glycogen synthase in which Lys15 is replaced by Gln via site-directed mutagenesis is inactivated by adenosine diphosphopyridoxal at concentrations higher than those required for the inactivation of the wild-type enzyme. ADP and ADP-glucose offered protective effects on inactivation, suggesting that the label binds to the ADP-glucose-binding site in the mutant enzyme. Sequence analysis of the labeled peptide revealed that the labeled residue is Lys277. This lysyl residue is conserved in maize starch synthase, which shows about 30% amino acid identity to E. coli glycogen synthase. Substitution of Gln for Lys277 by site-directed mutagenesis resulted in a 140-fold decrease in the kcat value with little changes in the Km values for ADP-glucose and glycogen. These results suggest that Lys277 at the active site participates in the catalytic reaction rather than binding of substrate. The present study shows the usefulness of the combined application of affinity labeling and site-directed mutagenesis.
authors.affiliation: Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Furukawa
authors.forename: M
authors.initials: M
authors.lastname: Tagaya
authors.forename: K
authors.initials: K
authors.lastname: Tanizawa
authors.forename: T
authors.initials: T
authors.lastname: Fukui
mesh_terms: D000345:Affinity Labels; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D006006:Glycogen Synthase / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
chemical_list: D000345:Affinity Labels; D017931:DNA Primers; D006006:Glycogen Synthase; D008239:Lysine