Kinetic and mutational analyses of the major cytosolic exopolyphosphatase from Saccharomyces cerevisiae

J Biol Chem. 2007 Mar 30;282(13):9302-9311. doi: 10.1074/jbc.M609423200. Epub 2007 Jan 10.

Abstract

Yeast exopolyphosphatase (scPPX) processively splits off the terminal phosphate group from linear polyphosphates longer than pyrophosphate. scPPX belongs to the DHH phosphoesterase superfamily and is evolutionarily close to the well characterized family II pyrophosphatase (PPase). Here, we used steady-state kinetic and binding measurements to elucidate the metal cofactor requirement for scPPX catalysis over the pH range 4.2-9.5. A single tight binding site for Mg(2+) (K(d) of 24 microm) was detected by equilibrium dialysis. Steady-state kinetic analysis of tripolyphosphate hydrolysis revealed a second site that binds Mg(2+) in the millimolar range and modulates substrate binding. This step requires two protonated and two deprotonated enzyme groups with pK(a) values of 5.0-5.3 and 7.6-8.2, respectively. The catalytic step requiring two deprotonated groups (pK(a) of 4.6 and 5.6) is modulated by ionization of a third group (pK(a) of 8.7). Conservative mutations of Asp(127), His(148), His(149) (conserved in scPPX and PPase), and Asn(35) (His in PPase) reduced activity by a factor of 600-5000. N35H and D127E substitutions reduced the Mg(2+) affinity of the tight binding site by 25-60-fold. Contrary to expectations, the N35H variant was unable to hydrolyze pyrophosphate, but markedly altered metal cofactor specificity, displaying higher catalytic activity with Co(2+) bound to the weak binding site versus the Mg(2+)- or Mn(2+)-bound enzyme. These results provide an initial step toward understanding the dynamics of scPPX catalysis and reveal significant functional differences between structurally similar scPPX and family II PPase.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid Anhydride Hydrolases / biosynthesis
  • Acid Anhydride Hydrolases / chemistry
  • Acid Anhydride Hydrolases / genetics*
  • Acid Anhydride Hydrolases / isolation & purification
  • Acid Anhydride Hydrolases / metabolism*
  • Amino Acid Sequence
  • Amino Acid Substitution / genetics*
  • Cytosol / enzymology*
  • Kinetics
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Sequence Analysis, Protein*
  • Sequence Homology, Amino Acid

Substances

  • Acid Anhydride Hydrolases
  • endopolyphosphatase
  • exopolyphosphatase