title: Directed mutagenesis studies of the C-terminal fingerprint region of Bacillus subtilis pyrophosphatase.
abstract: 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 pK(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.
authors.affiliation: Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Shizawa
authors.forename: T
authors.initials: T
authors.lastname: Uchiumi
authors.forename: J
authors.initials: J
authors.lastname: Taguchi
authors.forename: N A
authors.initials: NA
authors.lastname: Kisseleva
authors.forename: A A
authors.initials: AA
authors.lastname: Baykov
authors.forename: R
authors.initials: R
authors.lastname: Lahti
authors.forename: A
authors.initials: A
authors.lastname: Hachimori
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D011755:Pyrophosphatases