Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers.
Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg(2+)-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg(2+)-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer-trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.
Baykov
A A
AA
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Russia. baykov@genebee.msu.su
Hyytiä
T
T
Turkina
M V
MV
Efimova
I S
IS
Kasho
V N
VN
Goldman
A
A
Cooperman
B S
BS
Lahti
R
R
D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000378:metabolism*; D014325:Tromethamine
D014325:Tromethamine; D011755:Pyrophosphatases; D008274:Magnesium