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Identification and characterization of a critical region in the glycogen synthase from Escherichia coli.
Identification and characterization of a critical region in the glycogen synthase from Escherichia coli.
- Source :
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The Journal of biological chemistry [J Biol Chem] 2004 Feb 27; Vol. 279 (9), pp. 8359-67. Date of Electronic Publication: 2003 Dec 09. - Publication Year :
- 2004
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Abstract
- 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.
- Subjects :
- Adenosine Diphosphate Glucose metabolism
Adenosine Diphosphate Glucose pharmacology
Adenosine Monophosphate pharmacology
Amino Acid Sequence
Binding Sites
Computer Simulation
Conserved Sequence
Crystallization
Cysteine
Dithionitrobenzoic Acid pharmacology
Electrophoresis, Polyacrylamide Gel
Enzyme Inhibitors pharmacology
Gene Expression
Glucosephosphates pharmacology
Glycogen Synthase genetics
Glycogen Synthase metabolism
Iodoacetic Acid pharmacology
Kinetics
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Conformation
Structure-Activity Relationship
Substrate Specificity
Escherichia coli enzymology
Glycogen Synthase chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 279
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 14665620
- Full Text :
- https://doi.org/10.1074/jbc.M312686200