Effect of Asp-97->Glu Substitution on the pH Dependence of Catalysis by Inorganic Pyrophosphatase of Escherichia coli

I. P. Fabrichniy,¹ R. Lahti,² and A. A. Baykov^1,3

¹Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3181; E-mail: abaykov@protchem.genebee.msu.su

²Department of Biochemistry and Food Chemistry, University of Turku, Turku, FIN-20500, Finland; fax: (358-2) 633-6860; E-mail: reila@utu.fi

³To whom correspondence should be addressed.

Submitted April 10, 1997; revision submitted May 26, 1997.

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Substitution of Glu for the evolutionarily conserved Asp-97 in the active site of Escherichia coli inorganic pyrophosphatase increases the apparent pK_a of the essential acidic group controlling the catalytic constant for pyrophosphate hydrolysis. In combination with previously reported data, this fact suggests that activity decreases in alkaline medium because of decreased rate of the release of the first molecule of the product phosphate from the active site.

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KEY WORDS: pyrophosphatase, site-directed mutagenesis, pH dependence, kinetics.

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