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Effect of D42N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity and Mg 2+ binding
Author(s) -
Avaeva Svetlana M.,
Rodina Elena V.,
Kurilova Svetlana A.,
Nazarova Tatjana I.,
Vorobyeva Natalya N.
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 - 10.1016/0014-5793(96)00791-0
Subject(s) - inorganic pyrophosphatase , pyrophosphate , chemistry , pyrophosphatase , substrate (aquarium) , binding site , active center , hydrolysis , enzyme , escherichia coli , stereochemistry , pyrophosphatases , active site , enzyme kinetics , biochemistry , biology , ecology , gene
Asp‐42 located in the active site of E. coli inorganic pyrophosphatase (PPase) has been substituted by Asn by site‐directed mutagenesis. This resulted in a 3‐fold increase in hydrolytic activity measured under optimal conditions, a 15.5‐fold increase in the K m value and retention of the p K values of groups for enzyme and enzyme‐substrate complex. The active site of the enzyme contains 4 metal binding centers (I–IV) [Harutyunyan et al. (1996) Eur. J. Biochem., in press]. Asp‐42 is located near centers II and IV. The D42N replacement had no effect on Mg 2+ binding with center II. At the same time, occupation of center IV eliminates the inhibition of inorganic pyrophosphate hydrolysis by high Mg 2+ concentrations typical of wild‐type PPase. It is proposed that the increase in activity and decrease in affinity for substrate of the D42N PPase results from changes in Mg 2+ binding to center IV. The Mg 2+ binding centers of E. coli PPase are lined up in filling order.