Bacillus subtilis inorganic pyrophosphatase: the C-terminal signature sequence is essential for enzyme activity and conformational integrity.
Bacillus subtilis inorganic pyrophosphatase is the first member of a newly identified Family II of PPases. To examine the role of a signature sequence found near the C-terminus, two truncated variants and a series of site-specific mutants were produced. A truncation of 17 residues (17AATR) but also single alanine substitutions, R295A and K296A, produced inactive enzyme. Removal of 5 nonconserved terminal residues (5AATR) markedly affected enzyme stability. Replacing S294 with A, T, C, or V decreased activity, the latter two mutations showing the greatest effect. Substitutions V299I and V300I had no or minor effects, whereas V300W and V299G/V300W significantly reduced activity. The sizes of truncated proteins and the full-length PPase were indistinguishable by gel-filtration. We conclude that the C-terminus has no role in multimerization, while both its conserved and nonconserved regions are essential for full enzyme activity. The signature sequence is required for both the conformation and composition of the active site.
Konopka
Monika A
MA
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
White
Scott A
SA
Young
Tom W
TW
D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites / Q000502:physiology; D002850:Chromatography, Gel; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D043564:Inorganic Pyrophosphatase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase