Engineering a new magnesium binding site in the subunit contact region of Escherichia coli inorganic pyrophosphatase.
Three Gln-80 residues belonging to different subunits of homohexameric Escherichia coli pyrophosphatase are separated by only one water molecule to which they are hydrogen bonded. Substitution of Glu for Gln-80 stabilizes quaternary structure of the enzyme but has only a small effect on enzyme activity. The substitution stimulates Mg2+ binding and changes the appearance of the Mg2+ concentration dependence of the rate constant for the trimer --> hexamer transition. These data suggest that a new Mg2+ binding site is formed in the intersubunit contact region as a result of the substitution. Three-dimensional modeling of the mutated protein showed that a chelate complex might form involving two of the three Glu-80 residues.
Parfenyev
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AN
Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Lomonosov Moscow State University, Moscow, 119899, Russia.
Salminen
A
A
Baykov
A A
AA
Lahti
R
R
D019943:Amino Acid Substitution; D001665:Binding Sites; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005973:Glutamine / Q000378:metabolism; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011108:Polymers; D015202:Protein Engineering; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011756:Diphosphates; D011108:Polymers; D005973:Glutamine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium