Enhancement of the Rate of Pyrophosphate Hydrolysis by Nonenzymatic Catalysts and by Inorganic Pyrophosphatase

To estimate the proficiency of inorganic pyrophosphatase as a catalyst, 31P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PPi) in the presence and absence of Mg2+ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPPi2− proceeds with a rate constant of 2.8 × 10−10 s−1, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s−1 under the same conditions. The resulting rate enhancement (2 × 1012-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH‡ = −16.6 kcal/mol). The presence of Mg2+ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼106-fold. Transfer to dimethyl sulfoxide accelerated PPi hydrolysis by reducing the enthalpy of activation. Mg2+ increased the rate of PPi hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation. To estimate the proficiency of inorganic pyrophosphatase as a catalyst, 31P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PPi) in the presence and absence of Mg2+ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPPi2− proceeds with a rate constant of 2.8 × 10−10 s−1, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s−1 under the same conditions. The resulting rate enhancement (2 × 1012-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH‡ = −16.6 kcal/mol). The presence of Mg2+ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼106-fold. Transfer to dimethyl sulfoxide accelerated PPi hydrolysis by reducing the enthalpy of activation. Mg2+ increased the rate of PPi hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation. IntroductionEnzymes may have evolved from simpler catalysts that lowered the enthalpy of activation (ΔH‡) of biologically important reactions in the primitive earth. Thus, it is of interest to compare the thermodynamic basis of the rate enhancements produced by enzymes with the thermodynamic basis of the rate enhancements produced by small inorganic catalysts (1Stockbridge R.B. Lewis Jr., C.A. Yuan Y. Wolfenden R. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 22102-22105Crossref PubMed Scopus (43) Google Scholar).It is reasonable to suppose that inorganic pyrophosphate (PPi) 2The abbreviations used are: PPi, inorganic pyrophosphate; PPase, inorganic pyrophosphatase; DMSO, dimethyl sulfoxide. served as a phosphoryl group donor in primitive biological systems (2Baltscheffsky H. Lundin M. Luxemburg C. Nyren P. Baltscheffsky M. Chem. Scr. 1986; 26B: 259-262Google Scholar). Even today, PPi-dependent proton pumps, sodium pumps, and kinases are found in some bacteria (3Hedlund J. Cantoni R. Baltscheffsky M. Baltscheffsky H. Persson B. FEBS J. 2006; 273: 5183-5193Crossref PubMed Scopus (13) Google Scholar, 4Malinen A.M. Belogurov G.A. Baykov A.A. Lahti R. Biochemistry. 2007; 46: 8872-8878Crossref PubMed Scopus (47) Google Scholar, 5Ronimus R.S. Morgan H.W. Extremophiles. 2001; 5: 357-373Crossref PubMed Scopus (46) Google Scholar). PPi hydrolysis plays an important role in the metabolism of modern organisms by removing the PPi that is generated by biosynthetic reactions such as the synthesis of aminoacyl-tRNA. The enzyme (inorganic pyrophosphatase (PPase)) that catalyzes this reaction has an unusual active site that has been termed “mini-mineral,” in that metal ions and structural water molecules appear to be largely responsible for catalysis (6Cooperman B.S. Baykov A.A. Lahti R. Trends Biochem. Sci. 1992; 17: 262-266Abstract Full Text PDF PubMed Scopus (181) Google Scholar). This enzyme furnishes an opportunity to compare the proficiency of an enzyme that catalyzes one of the simpler reactions in biology with the proficiencies of primitive catalysts that model some of the key chemical features of the modern enzyme's active site.Two classes of PPases have been identified, and the tertiary structures of Type I PPases (found in eukaryotes and Escherichia coli) differ significantly from those of Type II PPases (found in some bacteria), suggesting that these two classes evolved independently (7Shintani T. Uchiumi T. Yonezawa T. Salminen A. Baykov A.A. Lahti R. Hachimori A. FEBS Lett. 1998; 439: 263-266Crossref PubMed Scopus (87) Google Scholar, 8Merckel M.C. Fabrichniy I.P. Salminen A. Kalkkinen N. Baykov A.A. Lahti R. Goldman A. Structure. 2001; 9: 289-297Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), yet these enzymes have remarkably similar active sites (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Type I PPases require three Mg2+ ions for activity (10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), whereas Type II PPases require four Mn2+ ions (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Metal ions and water molecules, organized and positioned by the protein, constitute the immediate environment of the bound substrate and have been proposed to assist catalysis in two ways (10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). First, the extremely polar active site neutralizes the charged PPi substrate so that the nucleophile can approach. A crystal structure of the Saccharomyces cerevisiae Type I enzyme complex with two bound phosphate ions shows that the oxygen atoms of the two phosphate groups are coordinated by six hydrogen bonds to water, six bonds to the metal ions, and six hydrogen bonds to protein side chains (10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In the Bacillus subtilis Type II enzyme with the substrate analog imidodiphosphate, all three nonbridging oxygen atoms of the phosphoryl group that undergoes attack by water are bound to a Mn2+ ion (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Second, metal ions coordinate the substrate water molecule, decreasing its pKa value and rendering it more effective as a nucleophile at ordinary pH values. In Type I PPases, the substrate water molecule bridges two metal ions (10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), whereas in Type II PPases, the substrate water molecule resides in the center of a plane formed by three metal ions (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).In the active sites of PPases, metal ions seem to stabilize the transition state to a greater extent than do protein side chains. Of the 14 conserved residues shared by Type I PPases, only two aspartic acid residues decreased kcat by more than ∼100-fold when mutated to alanine, and both of those mutations eliminated binding of the two structural Mg2+ ions (11Pohjanjoki P. Lahti R. Goldman A. Cooperman B.S. Biochemistry. 1998; 37: 1754-1761Crossref PubMed Scopus (34) Google Scholar). A water molecule appears to act as a general acid by protonating the leaving phosphoryl group (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Thus, PPase may constitute an extreme case in which metal ions and water molecules play a direct role in catalysis, whereas the protein moiety serves mainly as a scaffold for organizing the system.In principle, it does not seem obvious that a protein-based active site could not accelerate PPi hydrolysis. Kinases and phosphatases both require metal ions (generally one and two, respectively), yet these enzymes (which are structurally unrelated to PPases) employ protein side chains as catalytic bases, nucleophiles, or electrostatic catalysts (12Knowles J.R. Annu. Rev. Biochem. 1980; 49: 877-919Crossref PubMed Scopus (910) Google Scholar). The apparently convergent evolution of Type I and II PPases suggests that, in this special case, a primitive mini-mineral catalytic site might suffice to catalyze PPi hydrolysis.In this work, we sought to determine the magnitude and thermodynamic origin of the rate enhancements produced by E. coli PPase and by simple inorganic catalyst models of the enzyme's inorganic core. We compared the intrinsic reactivities of PPi4−, PPi3−, PPi2−, and MgPPi2− (the true substrate for PPase) (13Cooperman B.S. Methods Enzymol. 1982; 87: 526-548Crossref PubMed Scopus (100) Google Scholar). We also determined the rate constants and activation parameters for the hydrolysis of PPi by E. coli PPase. In addition, we examined the rate of PPi hydrolysis in dimethyl sulfoxide (DMSO), a dipolar aprotic solvent chosen to explore the effect of transferring this substrate from solvent water to a somewhat polar nonaqueous environment. IntroductionEnzymes may have evolved from simpler catalysts that lowered the enthalpy of activation (ΔH‡) of biologically important reactions in the primitive earth. Thus, it is of interest to compare the thermodynamic basis of the rate enhancements produced by enzymes with the thermodynamic basis of the rate enhancements produced by small inorganic catalysts (1Stockbridge R.B. Lewis Jr., C.A. Yuan Y. Wolfenden R. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 22102-22105Crossref PubMed Scopus (43) Google Scholar).It is reasonable to suppose that inorganic pyrophosphate (PPi) 2The abbreviations used are: PPi, inorganic pyrophosphate; PPase, inorganic pyrophosphatase; DMSO, dimethyl sulfoxide. served as a phosphoryl group donor in primitive biological systems (2Baltscheffsky H. Lundin M. Luxemburg C. Nyren P. Baltscheffsky M. Chem. Scr. 1986; 26B: 259-262Google Scholar). Even today, PPi-dependent proton pumps, sodium pumps, and kinases are found in some bacteria (3Hedlund J. Cantoni R. Baltscheffsky M. Baltscheffsky H. Persson B. FEBS J. 2006; 273: 5183-5193Crossref PubMed Scopus (13) Google Scholar, 4Malinen A.M. Belogurov G.A. Baykov A.A. Lahti R. Biochemistry. 2007; 46: 8872-8878Crossref PubMed Scopus (47) Google Scholar, 5Ronimus R.S. Morgan H.W. Extremophiles. 2001; 5: 357-373Crossref PubMed Scopus (46) Google Scholar). PPi hydrolysis plays an important role in the metabolism of modern organisms by removing the PPi that is generated by biosynthetic reactions such as the synthesis of aminoacyl-tRNA. The enzyme (inorganic pyrophosphatase (PPase)) that catalyzes this reaction has an unusual active site that has been termed “mini-mineral,” in that metal ions and structural water molecules appear to be largely responsible for catalysis (6Cooperman B.S. Baykov A.A. Lahti R. Trends Biochem. Sci. 1992; 17: 262-266Abstract Full Text PDF PubMed Scopus (181) Google Scholar). This enzyme furnishes an opportunity to compare the proficiency of an enzyme that catalyzes one of the simpler reactions in biology with the proficiencies of primitive catalysts that model some of the key chemical features of the modern enzyme's active site.Two classes of PPases have been identified, and the tertiary structures of Type I PPases (found in eukaryotes and Escherichia coli) differ significantly from those of Type II PPases (found in some bacteria), suggesting that these two classes evolved independently (7Shintani T. Uchiumi T. Yonezawa T. Salminen A. Baykov A.A. Lahti R. Hachimori A. FEBS Lett. 1998; 439: 263-266Crossref PubMed Scopus (87) Google Scholar, 8Merckel M.C. Fabrichniy I.P. Salminen A. Kalkkinen N. Baykov A.A. Lahti R. Goldman A. Structure. 2001; 9: 289-297Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), yet these enzymes have remarkably similar active sites (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Type I PPases require three Mg2+ ions for activity (10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), whereas Type II PPases require four Mn2+ ions (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Metal ions and water molecules, organized and positioned by the protein, constitute the immediate environment of the bound substrate and have been proposed to assist catalysis in two ways (10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). First, the extremely polar active site neutralizes the charged PPi substrate so that the nucleophile can approach. A crystal structure of the Saccharomyces cerevisiae Type I enzyme complex with two bound phosphate ions shows that the oxygen atoms of the two phosphate groups are coordinated by six hydrogen bonds to water, six bonds to the metal ions, and six hydrogen bonds to protein side chains (10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In the Bacillus subtilis Type II enzyme with the substrate analog imidodiphosphate, all three nonbridging oxygen atoms of the phosphoryl group that undergoes attack by water are bound to a Mn2+ ion (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Second, metal ions coordinate the substrate water molecule, decreasing its pKa value and rendering it more effective as a nucleophile at ordinary pH values. In Type I PPases, the substrate water molecule bridges two metal ions (10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), whereas in Type II PPases, the substrate water molecule resides in the center of a plane formed by three metal ions (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).In the active sites of PPases, metal ions seem to stabilize the transition state to a greater extent than do protein side chains. Of the 14 conserved residues shared by Type I PPases, only two aspartic acid residues decreased kcat by more than ∼100-fold when mutated to alanine, and both of those mutations eliminated binding of the two structural Mg2+ ions (11Pohjanjoki P. Lahti R. Goldman A. Cooperman B.S. Biochemistry. 1998; 37: 1754-1761Crossref PubMed Scopus (34) Google Scholar). A water molecule appears to act as a general acid by protonating the leaving phosphoryl group (9Fabrichniy I.P. Lehtiö L. Tammenkoski M. Zyryanov A.B. Oksanen E. Baykov A.A. Lahti R. Goldman A. J. Biol. Chem. 2007; 282: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Heikinheimo P. Lehtonen J. Baykov A. Lahti R. Cooperman B.S. Goldman A. Structure. 1996; 4: 1491-1508Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Thus, PPase may constitute an extreme case in which metal ions and water molecules play a direct role in catalysis, whereas the protein moiety serves mainly as a scaffold for organizing the system.In principle, it does not seem obvious that a protein-based active site could not accelerate PPi hydrolysis. Kinases and phosphatases both require metal ions (generally one and two, respectively), yet these enzymes (which are structurally unrelated to PPases) employ protein side chains as catalytic bases, nucleophiles, or electrostatic catalysts (12Knowles J.R. Annu. Rev. Biochem. 1980; 49: 877-919Crossref PubMed Scopus (910) Google Scholar). The apparently convergent evolution of Type I and II PPases suggests that, in this special case, a primitive mini-mineral catalytic site might suffice to catalyze PPi hydrolysis.In this work, we sought to determine the magnitude and thermodynamic origin of the rate enhancements produced by E. coli PPase and by simple inorganic catalyst models of the enzyme's inorganic core. We compared the intrinsic reactivities of PPi4−, PPi3−, PPi2−, and MgPPi2− (the true substrate for PPase) (13Cooperman B.S. Methods Enzymol. 1982; 87: 526-548Crossref PubMed Scopus (100) Google Scholar). We also determined the rate constants and activation parameters for the hydrolysis of PPi by E. coli PPase. In addition, we examined the rate of PPi hydrolysis in dimethyl sulfoxide (DMSO), a dipolar aprotic solvent chosen to explore the effect of transferring this substrate from solvent water to a somewhat polar nonaqueous environment.