Biochemical Journal

Research article

Mechanism by which metal cofactors control substrate specificity in pyrophosphatase

Anton B. ZYRYANOV, Alexander S. SHESTAKOV, Reijo LAHTI, Alexander A. BAYKOV


Family I soluble pyrophosphatases (PPases) exhibit appreciable ATPase activity in the presence of a number of transition metal ions, but not the physiological cofactor Mg2+. The results of the present study reveal a strong correlation between the catalytic efficiency of three family I PPases (from Saccharomyces cerevisiae, Escherichia coli and rat liver) and one family II PPase (from Streptococcus mutans) in ATP and tripolyphosphate (P3) hydrolysis in the presence of Mg2+, Mn2+, Zn2+ and Co2+ on the one hand, and the phosphate-binding affinity of the enzyme subsite P2 that interacts with the electrophilic terminal phosphate group of ATP on the other. A similar correlation was observed in S. cerevisiae PPase variants with modified P1 and P2 subsites. The effect of the above metal ion cofactors on ATP binding to S. cerevisiae PPase paralleled their effect on phosphate binding, resulting in a low affinity of Mg-PPase to ATP. We conclude that PPase mainly binds ATP and P3 through the terminal phosphate group that is attacked by water. Moreover, this interaction is critical in creating a reactive geometry at the P2 site with these bulky substrates, which do not otherwise fit the active site perfectly. We propose further that ATP is not hydrolysed by Mg-PPase, since its interaction with the terminal phosphate is not adequately strong for proper positioning of the nucleophile—electrophile pair.

  • ATP
  • ATPase
  • Pi
  • tripolyphosphate


  • Abbreviations used: K56R etc., Lys56→Arg etc.; PPase, pyrophosphatase; E-PPase, PPase isolated from Escherichia coli; R-PPase, PPase isolated from rat liver; Sm-PPase, PPase isolated from Streptococcus mutans; Y-PPase, PPase isolated from Saccharomyces cerevisiae; P3, tripolyphosphate; R78K, Arg78→Lys; Y93F, Tyr93→Phe.