2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: firstname.lastname@example.org
3Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 119333 Moscow, Russia; E-mail: email@example.com
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Received December 23, 2008; Revision received February 12, 2009
In this paper, kinetic properties of a soluble inorganic pyrophosphatase of family I from Vibrio cholerae (V-PPase), intestinal pathogen and causative agent of human cholera, are characterized in detail, and the crystal structure of a metal-free enzyme is reported. Hydrolytic activity of V-PPase has been studied as a function of pH, concentration of metal cofactors (Mg2+ or Mn2+), and ionic strength. It has been found that, despite the high conservation of amino acid sequences for the known bacterial PPases of family I, V-PPase differs from the other enzymes of the same family in a number of parameters. Dissociation constants of V-PPase complexed with Mg2+ or Mn2+ were essentially the same as for Escherichia coli PPase (E-PPase). However, the pH optimum of MgPPi hydrolysis by V-PPase was shifted to more alkaline pH due to higher values of the pKa of ionizable groups for both the free enzyme and the enzyme–substrate complex. The stability of a hexameric form of V-PPase has been studied as a function of pH. The corresponding pKa of a group that controls the stability of the hexamer at pH below 6 (pKa = 4.4) was significantly lower than in the other hexameric PPases. The crystal structure reported here is analyzed and compared with the structure of E-PPase. The location of amino acid residues that differ in V-PPase and E-PPase is discussed. Since V-PPase has been found to retain its hydrolytic activity in high ionic strength media, the observed structural and kinetic features are analyzed in view of the possible osmoadaptation of this protein.
KEY WORDS: pyrophosphatase, Vibrio cholerae, osmoadaptation, X-ray analysis, crystal structure