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J. Biol. Chem., Vol. 282, Issue 13, 9302-9311, March 30, 2007

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Kinetic and Mutational Analyses of the Major Cytosolic Exopolyphosphatase from Saccharomyces cerevisiae^*

Marko Tammenkoski, Viktor M. Moiseev, Matti Lahti, Emilie Ugochukwu^¶, T. Harma C. Brondijk^¶, Scott A. White^¶, Reijo Lahti¹, and Alexander A. Baykov²

From the Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland, the A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia, and the ^¶School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

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 Mg²⁺ (K_d of 24 µM) was detected by equilibrium dialysis. Steady-state kinetic analysis of tripolyphosphate hydrolysis revealed a second site that binds Mg²⁺ 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 Asp¹²⁷, His¹⁴⁸, His¹⁴⁹ (conserved in scPPX and PPase), and Asn³⁵ (His in PPase) reduced activity by a factor of 600–5000. N35H and D127E substitutions reduced the Mg²⁺ 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 Co²⁺ bound to the weak binding site versus the Mg²⁺- or Mn²⁺-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.

Received for publication, October 5, 2006 , and in revised form, January 8, 2007.

^* This work was supported in part by grants from the Academy of Finland (Nos. 201611 and 114706), the Russian Foundation for Basic Research (No. 06-04-48887), the Ministry of Education, and the Academy of Finland (for the National Graduate School in Informational and Structural Biology), and the Finnish Cultural Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. Tel.: 358-2-333-6845; Fax: 358-2-333-6860; E-mail: reijo.lahti{at}utu.fi. ² To whom correspondence may be addressed. Tel.: 7-495-939-5541; Fax: 7-495-939-3181; E-mail: baykov{at}genebee.msu.su.

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