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.
Highlights
Characterization of Yeast Exopolyphosphatase are very similar, parts of the N-terminal domains (Fig. 1B). Despite these structural and functional similarities, the substrate specificities of structure of yeast cytosolic PPX (scPPX) and family II PPase are opposite. scPPX is highly active against linear polyphosphates formed by three or more phosphate residues but inactive against pyrophosphate [19, 20], whereas family II PPase is active against pyrophosphate but displays low activity against longer polyphosphates [27]
The results provide an initial step toward understanding the dynamics of scPPX catalysis
Expression and Purification of scPPX—SDS-PAGE analysis of crude extracts obtained from recombinant E. coli cells revealed an intense 45-kDa band corresponding to yeast PPX subunit [12] and a very faint band of 58 kDa representing E. coli PPX [10]
Summary
Are very similar, parts of the N-terminal domains (Fig. 1B). Despite these structural and functional similarities, the substrate specificities of scPPX and family II PPase are opposite. scPPX is highly active against linear polyphosphates formed by three or more phosphate residues but inactive against pyrophosphate [19, 20], whereas family II PPase is active against pyrophosphate but displays low activity against longer polyphosphates [27]. ScPPX is highly active against linear polyphosphates formed by three or more phosphate residues but inactive against pyrophosphate [19, 20], whereas family II PPase is active against pyrophosphate but displays low activity against longer polyphosphates [27] This difference in specificity may arise from scPPX having an extended substrate binding site. Steady-state kinetic and binding measurements were applied to determine the stoichiometry of Mg2ϩ participation at different steps of catalysis by scPPX within the pH range 4.2–9.5. These experiments yielded information on the ionizing groups involved in metal binding and catalysis. The results provide an initial step toward understanding the dynamics of scPPX catalysis
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