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Contribution of the two domains of E. coli 5′‐nucleotidase to substrate specificity and catalysis
Author(s) -
Krug Ulrike,
Patzschke Rica,
Zebisch Matthias,
Balbach Jochen,
Sträter Norbert
Publication year - 2013
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2013.01.010
Subject(s) - escherichia coli , nucleotide , substrate (aquarium) , chemistry , atp hydrolysis , catalysis , hydrolysis , phosphate , enzyme , nucleotidase , stereochemistry , biochemistry , biology , ecology , atpase , gene
Escherichia coli 5′‐nucleotidase, a two‐domain enzyme, dephosphorylates various nucleotides with comparable efficiency. We have expressed the two domains individually in E. coli and show by liquid state NMR that they are properly folded. Kinetic characterization reveals that the C‐terminal domain, which contains the substrate‐binding pocket, is completely inactive while the N‐terminal domain with the two‐metal‐ion‐center and the core catalytic residues exhibits significant activity, especially towards substrates with activated phosphate bonds (ATP, ADP, p‐nitrophenyl phosphate). In contrast, residues of the C‐terminal domain are required for efficient hydrolysis of AMP.