Open AccessDirected mutagenesis studies of the C‐terminal fingerprint region of Bacillus subtilis pyrophosphatase
Author(s) -
Shizawa Nobuko,
Uchiumi Toshio,
Taguchi Junko,
Kisseleva Natalya A.,
Baykov Alexander A.,
Lahti Reijo,
Hachimori Akira
Publication year - 2001
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.0014-2956.2001.02513.x
Subject(s) - pyrophosphatases , bacillus subtilis , pyrophosphatase , mutagenesis , enzyme , inorganic pyrophosphatase , substrate (aquarium) , biochemistry , active site , chemistry , stereochemistry , biology , crystallography , mutation , bacteria , genetics , ecology , gene , pyrophosphate
The sequence SRKKQxxP near the C‐terminus is conserved in pyrophosphatases of the recently discovered family II and includes a triplet of positively charged residues, two of which (Arg295 and Lys296 in Bacillus subtilis pyrophosphatase) are part of the active site and one (Lys297) is not. The importance of this triplet for catalysis by B. subtilis pyrophosphatase has been estimated by mutational analysis. R295K and K296R substitutions were found to decrease the catalytic constant 650‐ and 280‐fold, respectively, and decrease the p K a of the essential acidic group by 1.1 and 0.5, respectively. K297R substitution was found to increase the catalytic constant 4.7‐fold and to markedly change the protein circular dichroism spectrum in the range 250–300 nm. These results, together with the results of theoretical modelling of the enzyme–substrate complex, provide support for the direct involvement of Arg295 and Lys296 in substrate binding in family II pyrophosphatases.