Identification and characterization of a critical region in the glycogen synthase from Escherichia coli.
The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding.
Yep
Alejandra
A
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Ballicora
Miguel A
MA
Sivak
Mirta N
MN
Preiss
Jack
J
D000245:Adenosine Diphosphate Glucose / Q000378:metabolism / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D017124:Conserved Sequence; D003460:Crystallization; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D005958:Glucosephosphates / Q000494:pharmacology; D006006:Glycogen Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019807:Iodoacetic Acid / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005958:Glucosephosphates; D000245:Adenosine Diphosphate Glucose; D000249:Adenosine Monophosphate; D004228:Dithionitrobenzoic Acid; C031590:glucose-1-phosphate; D006006:Glycogen Synthase; D003545:Cysteine; D019807:Iodoacetic Acid