TY - JOUR
T1 - Bacillus subtilis inorganic pyrophosphatase: The C-terminal signature sequence is essential for enzyme activity and conformational integrity
AU - Konopka, Monika
AU - White, Scott
AU - Young, Thomas
PY - 2002/1/18
Y1 - 2002/1/18
N2 - 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. (C) 2002 Elsevier Science.
AB - 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. (C) 2002 Elsevier Science.
KW - inorganic pyrophosphatase
KW - signature sequence
KW - Bacillus subtilis
KW - site-directed mutagenesis
KW - C-terminal truncation
UR - http://www.scopus.com/inward/record.url?scp=0036291155&partnerID=8YFLogxK
U2 - 10.1006/bbrc.2001.6250
DO - 10.1006/bbrc.2001.6250
M3 - Article
VL - 290
SP - 806
EP - 812
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -