2Department of Biochemistry, University of Turku, Turku, FIN-20500, Finland; fax: (358-2) 633-6860; E-mail: firstname.lastname@example.org
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Received October 12, 1999
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.
KEY WORDS: pyrophosphatase, site-directed mutagenesis, magnesium