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Stabilization of the Enzyme--Substrate Complex of the Mutant Asp-67Asn Inorganic Pyrophosphatase from Escherichia coli by Fluoride Ions

S. M. Avaeva1*, T. I. Velichko2, N. N. Vorobyeva2, S. A. Kurilova1, T. I. Nazarova1, and V. A. Sklyankina2

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

2School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia

* To whom correspondence should be addressed.

Received June 9, 1998; Revision received June 26, 1998
Magnesium-supported PPi hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PPi and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PPi hydrolysis. The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly. This enzyme--substrate complex stabilized by fluoride is partially active and can be isolated by the removal of excess fluoride by gel-filtration.
KEY WORDS: inorganic pyrophosphatase, Escherichia coli, inhibition by fluoride, enzyme--substrate complex