ATP as Effector of Inorganic Pyrophosphatase of Escherichia coli. Identification of the Binding Site for ATP

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E. V. Rodina^1,2*, N. N. Vorobyeva¹, S. A. Kurilova², M. S. Belenikin¹, N. V. Fedorova², and T. I. Nazarova²

¹Chemical Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: rodina@belozersky.msu.ru

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: nazarova@belozersky.msu.ru

^* To whom correspondence should be addressed.

Received August 1, 2006; Revision received September 6, 2006

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The interaction of Escherichia coli inorganic pyrophosphatase (E-PPase) with effector ATP has been studied. The E-PPase has been chemically modified with the dialdehyde derivative of ATP. It has been established that in the experiment only one molecule of effector ATP is bound to each subunit of the hexameric enzyme. Tryptic digestion of the adenylated protein followed by isolation of a modified peptide by HPLC and its mass-spectrometric identification has showed that it is an amino group of Lys146 that undergoes modification. Molecular docking of ATP to E-PPase indicates that the binding site for effector ATP is located in a cluster of positively charged amino acid residues proposed earlier on the basis of site-directed mutagenesis to participate in binding of effector pyrophosphate. Molecular docking also reveals several other amino acid residues probably involved in the interaction with effectors.

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KEY WORDS: pyrophosphatase, ATP, binding site, chemical modification, MALDI-TOF, molecular docking

DOI: 10.1134/S0006297907010117

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