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

S. M. Avaeva^1*, 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

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Magnesium-supported PP_i hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PP_i and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PP_i 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.

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KEY WORDS: inorganic pyrophosphatase, Escherichia coli, inhibition by fluoride, enzyme--substrate complex

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