, Volume 70, Issue 8, pp 858-866

Substitutions of Glycine Residues Gly100 and Gly147 in Conservative Loops Decrease Rates of Conformational Rearrangements of Escherichia coli Inorganic Pyrophosphatase

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Escherichia coli inorganic pyrophosphatase (PPase) is a one-domain globular enzyme characterized by its ability to easily undergo minor structure rearrangements involving flexible segments of the polypeptide chain. To elucidate a possible role of these segments in catalysis, catalytic properties of mutant variants of E. coli PPase Gly100Ala and Gly147Val with substitutions in the conservative loops II and III have been studied. The main result of the mutations was a sharp decrease in the rates of conformational changes required for binding of activating Mg2+ ions, whereas affinity of the enzyme for Mg2+ was not affected. The pH-independent parameters of MgPPi hydrolysis, k cat and k cat/K m, have been determined for the mutant PPases. The values of k cat for Gly100Ala and Gly147Val variants were 4 and 25%, respectively, of the value for the native enzyme. Parameter k cat/K m for both mutants was two orders of magnitude lower. Mutation Gly147Val increased pH-independent K m value about tenfold. The study of synthesis of pyrophosphate in the active sites of the mutant PPases has shown that the maximal level of synthesized pyrophosphate was in the case of Gly100Ala twofold, and in the case of Gly147Val fivefold, higher than for the native enzyme. The results reported in this paper demonstrate that the flexibility of the loops where the residues Gly100 and Gly147 are located is necessary at the stages of substrate binding and product release. In the case of Gly100Ala PPase, significant impairment of affinity of enzyme effector site for PPi was also found.

Translated from Biokhimiya, Vol. 70, No. 8, 2005, pp. 1041–1050.
Original Russian Text Copyright © 2005 by Moiseev, Rodina, Kurilova, Vorobyeva, Nazarova, Avaeva.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM04-049, July 24, 2005.