Binding of Substrate at the Effector Site of Pyrophosphatase Increases the Rate of Its Hydrolysis at the Active Site

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T. S. Sitnik and S. M. Avaeva^*

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: avaeva@belozersky.msu.ru

^* To whom correspondence should be addressed.

Received June 30, 2006; Revision received September 11, 2006

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It is shown that in addition to the active site, each subunit of Escherichia coli inorganic pyrophosphatase (E-PPase) contains an extra binding site for the substrate magnesium pyrophosphate or its non-hydrolyzable analog magnesium methylenediphosphonate. The occupancy of the extra site stimulates the substrate conversion. Binding affinity of this site decreased or disappeared upon the conversion of E-PPase into a trimeric form or introduction of point mutations. However, when the slowly hydrolyzed substrate, lanthanum pyrophosphate (LaPP_i), is used, the extra site was revealed in all enzyme forms of E-PPase and of Y-PPase (Saccharomyces cerevisiae PPase), resulting in about 100-fold activation of hydrolysis. A hypothesis on the localization of the extra site and the mechanism of its effect in E-PPase is presented.

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KEY WORDS: inorganic pyrophosphatase, effector site, activation by substrate

DOI: 10.1134/S0006297907010087

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