Escherichia coli inorganic pyrophosphatase: site-directed mutagenesis of the metal binding sites.
Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine. Each mutation was found to decrease the k(app) value by approx. 2-3 orders of magnitude. At the same time, the Km values changed only slightly. Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis. All mutant variants have practically the same affinity to Mg2+ as the wild-type pyrophosphatase.
Avaeva
S
S
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russian Federation. avaeva@protchem.genebee.msu.su
Ignatov
P
P
Kurilova
S
S
Nazarova
T
T
Rodina
E
E
Vorobyeva
N
N
Oganessyan
V
V
Harutyunyan
E
E
D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D008274:Magnesium / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium