title: Effects of divalent cations on thermophilic inorganic pyrophosphatase.
abstract: Divalent cations were shown to affect the structure and thermostability of thermophilic inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] purified from Bacillus stearothermophilus and thermophilic bacterium PS-3. The properties of the enzymes from the two sources were found to be very similar. The enzymes were very unstable to heart in the absence of divalent cations, being inactivated gradually even at 40 degrees C. However, they became stable to heat denaturation in the presence of Mg2+, between pH 7.8 and 9.0. Similar induced thermostability was detected when Mn2+, Co2+, Ca2+, Cd2+, and ZN2+ were added, though the latter three cations were not essential for enzyme activity. On adding divalent cations, the optical properties such as absorption spectra, fluorescence spectra, and circular dichroism (CD) were changed. Gel filtration and disc electrophoresis revealed that the molecular weight of both enzymes was 5.4 x 10(4) in Tris-SO4 buffer and 11 x 10(4) in Tris-HCL buffer, suggesting monomer-dimer transformation. In the presence of divalent cations in Tris-SO4 fuffer, the enzymes dimerized; this was confirmed by sedimentation velocity measurements. The enzymes in Tris-HCL buffer did not show thermostability unless divalent cations were added. The results in the present study indicate that binding of divalent cations to each enzyme caused some conformational change in the vicinity of aromatic amino acid residues leading to dimerization of the enzyme molecule so that it became thermostable. It was also suggested that histidyl residues play an important role in the thermostability induced by divalent cations on the basis of the pH dependencies of thermostability and CD spectra.
mesh_terms: D001419:Bacteria / Q000201:enzymology*; D002413:Cations, Divalent; D002942:Circular Dichroism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism*; D013045:Species Specificity; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D046911:Macromolecular Substances; D011755:Pyrophosphatases