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Escherichia coli inorganic pyrophosphatase : site‐directed mutagenesis of the metal binding sites
Author(s) -
Avaeva Svetlana,
Ignatov Pavel,
Kurilova Svetlana,
Nazarova Tatjana,
Rodina Elena,
Vorobyeva Natalja,
Oganessyan Vaheh,
Harutyunyan Emil
Publication year - 1996
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/s0014-5793(96)01296-3
Subject(s) - inorganic pyrophosphatase , escherichia coli , asparagine , pyrophosphatase , site directed mutagenesis , mutagenesis , mutant , chemistry , binding site , biochemistry , aspartic acid , substrate (aquarium) , mutation , pyrophosphatases , active site , stereochemistry , enzyme , biology , amino acid , pyrophosphate , ecology , gene
Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli , identified as evolutionarily conserved residues of the active site, have been replaced by asparagine. Each mutation was found to decrease the κ app value by approx. 2–3 orders of magnitude. At the same time, the K m values changed only slightly. Only minor changes take place in the p K values of the residues essential for both substrate binding and catalysis. All mutant variants have practically the same affinity to Mg 2+ as the wild‐type pyrophosphatase.