Metal Cofactors Play a Dual Role in Mycobacterium tuberculosis Inorganic Pyrophosphatase

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E. V. Rodina^1,2*, L. P. Vainonen¹, N. N. Vorobyeva¹, S. A. Kurilova², T. S. Sitnik², and T. I. Nazarova²

¹Chemical Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 932-8846; 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 December 25, 2007; Revision received January 24, 2008

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Inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) is one of the possible targets for the rational design of anti-tuberculosis agents. In this paper, functional properties of this enzyme are characterized in the presence of the most effective activators--Mg²⁺ and Mn²⁺. Dissociation constants of Mt-PPase complexed with Mg²⁺ or Mn²⁺ are essentially similar to those of Escherichia coli PPase. Stability of a hexameric form of Mt-PPase has been characterized as a function of pH both for the metal-free enzyme and for Mg²⁺- or Mn²⁺-enzyme. Hexameric metal-free Mt-PPase has been shown to dissociate, forming monomers at pH below 4 or trimers at pH from 8 to 10. Mg²⁺ or Mn²⁺ shift the hexamer-trimer equilibrium found for the apo-Mt-PPase at pH 8-10 toward the hexameric form by stabilizing intertrimeric contacts. The pK_a values have been determined for groups that control the observed hexamer-monomer (pK_a 5.4), hexamer-trimer (pK_a 7.5), and trimer-monomer (pK_a 9.8) transitions. Our results demonstrate that due to the non-conservative amino acid residues His21 and His86 in the active site of Mt-PPase, substrate specificity of this enzyme, in contrast to other typical PPases, does not depend on the nature of the metal cofactor.

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KEY WORDS: pyrophosphatase, M. tuberculosis, Mg²⁺, Mn²⁺, metal cofactor, cofactor specificity

DOI: 10.1134/S0006297908080075

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