The kinetic mechanism of yeast inorganic pyrophosphatase

The kinetic mechanism of yeast inorganic pyrophosphatase (PPase) was examined by carrying out initial velocity studies. Ca2+ and Rh(H2O)4(methylenediphosphonate) (Rh(H2O)4PCP) were used as dead-end inhibitors to study the order of binding of Cr(H2O)4PP to the substrate site and Mg2+ to the "low affinity" activator site on the enzyme. Competitive inhibition was observed for Ca2+ vs Mg2+ (Kis = 0.93 +/- 0.03 mM), for Rh(H2O)4PCP vs Cr(H2O)4PP (Kis = 0.25 +/- 0.07 mM), and for RH(H2O)4PCP vs Mg2+ (Kis = 0.38 +/- 0.03 mM). Uncompetitive inhibition was observed for Ca2+ vs Cr(H2O)4PP (Kii = 0.49 +/- 0.01). On the basis of these results a rapid equilibrium ordered mechanism in which Cr(H2O)4PP binding precedes Mg2+ ion binding is proposed. The inert substrate analog, Mg(imidodiphosphate) (MgPNP) was shown to induce Mg2+ inhibition of the PPase-catalyzed hydrolysis of MgPP. The Mg2+ inhibition observed was competitive vs MgPP and partial. These results suggest that Mg2+/MgPNP release from the enzyme occurs in preferred rather than strict order and that the Mg2+/MgPP-binding steps are at steady state. Zn2+, Co2+, and Mn2+ (but not Mg2+) displayed activator inhibition of the PPase-catalyzed hydrolysis of PPi (this study) and of Cr(H2O)4PP (W.B. Knight, S. Fitts, and D. Dunaway-Mariano, (1981) Biochemistry 20, 4079). These findings suggest that cofactor release from the low affinity cofactor site on the enzyme must precede product release and that Zn2+, Mn2+, and Co2+ (but not Mg2+) have high affinities for the cofactor sites on both the PPase.M.MPP and PPase.M.M(P)2 complexes. The role of the metal cofactor in determining PPase substrate specificity was briefly explored by testing the ability of the Mg2+ complex of tripolyphosphate (PPPi) (a substrate for the Zn2+-activated enzyme but not the Mg2+-activated enzyme) to induce Mg2+ inhibition of PPase-catalyzed hydrolysis of MgPP. MgPPP was shown to be as effective as MgPNP in inducing competitive Mg2+ inhibition (vs MgPP). This result suggests that the low affinity Mg2+ cofactor-binding site present in the enzyme-MgPP complex is maintained in the enzyme-MgPPP complex. Thus, failure of Mg2+ to bind to the enzyme-MgPPP complex was ruled out as a possible explanation for the failure of the Mg2+-activated enzyme to catalyze the hydrolysis of MgPPP.     

Investigation of the catalytic mechanism of yeast inorganic pyrophosphatase

P1,P2-Bidentate Co(NH3)4PP was found to be a competitive inhibitor of pyrophosphatase vs. MgPP (Kis = 8.7 mM, pH 7) and, in the presence of Mg2+, an active substrate as well. P1,P2-Bidentate Cr(III) complexes of pyrophosphate, imidodiphosphate, and methylenediphosphonate were also competitive inhibitors vs. MgPP (pH 5.9; Kis = 0.2, 0.2, and 0.4 mM, respectively). In the presence of Mg2+, P1,P2-bidentate Cr(H2O)4PP was found to have a Km 10-fold greater and a turnover number 36-fold smaller than MgPP at pH 5.9. Mg2+, Mn2+, Co2+, Zn2+, Cd2+, Ni2+, and Fe2+ activate the CrPP--pyrophosphatase reaction, while Ca2+ and Ba2+ are not activators but serve as competitive inhibitors vs. Mg2+ (Kis = 0.35 and 2.3 mM). At levels above 0.1 mM, Mn2+, Co2+, and Zn2+ show activator inhibition. Kinetic studies with CrPP and Mg2+ suggest that the kinetic mechanism is rapid equilibrium ordered, with CrPP adding before Mg2+. pH studies of the MgPP/Mg2+ reaction and the CrPP/Mg2+ reaction suggest that the active form of the substrate is (MgPP)2- and that the uncomplexed metal ion cofactor interacts with at least two active-site residues, one possibly via H bonding and the other by direct coordination. The former group (pKa = 5.6) appears on the basis of temperature and solvent perturbation studies to be a carboxylic acid. The MgPP reaction also requires that an active-site residue (pKa = 7.5) be protonated. Temperature and solvent perturbation studies suggest that this residue is an amine. A mechanism accounting for these observations is presented.     

Cooperative mechanism of phosphorylation of the monomeric and dimeric forms of inorganic pyrophosphatase from baker's yeast

A comparative study of phosphorylation of native dimeric and artificial monomeric forms of inorganic pyrophosphatase and its fluoride-stabilized complex with PPi has been carried out. The maximal incorporation of Pi for the dimeric and monomeric proteins is 0.5 and 1 mole per mole of subunit, respectively. The saturation kinetic curves are suggestive of strong positive cooperative interactions. The value of the Hill coefficient (5.5) for the free dimeric enzyme drastically changes upon the active center blockage and/or transition to the monomeric enzyme. Acceleration of dephosphorylation induced by Pi in the presence of Mg2+ is observed only in the case of the dimeric protein. The data obtained indicate that phosphorylation of native dimeric pyrophosphatase occurs according to a "flip-flop" mechanism; the Pi binding in the active center exerts a strong influence on individual steps of the reaction.     

Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.     

Crystalline inorganic pyrophosphatase isolated from baker's yeast

Crystalline inorganic pyrophosphatase has been isolated from baker's yeast. The crystalline enzyme is a protein of the albumin type with an isoelectric point near pH 4.8. Its molecular weight is of the order of 100,000. It contains about 5 per cent tyrosine and 3.5 per cent tryptophane. It is most stable at pH 6.8. The new crystalline protein acts as a specific catalyst for the hydrolysis of inorganic pyrophosphate into orthophosphate ions. It does not catalyze the hydrolysis of the pyrophosphate radical of such organic esters as adenosine di- and triphosphate, or thiamine pyrophosphate. Crystalline pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions. The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of enzyme and is a function of pH, temperature, concentration of substrate, and concentration of activating ion. The approximate conditions for optimum rate are: 40 degrees C. and pH 7.0 at a concentration of 3 to 4 x 10(-3)M Na(4)P(2)O(7) and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of Na(4)P(2)O(7) or K(4)P(2)O(7) proceeds to completion and is irreversible under the conditions at which hydrolysis is occurring. Details are given of the method of isolation of the crystalline enzyme.     

Allosteric regulation of yeast inorganic pyrophosphatase by substrate

Kinetic and binding studies of yeast inorganic pyrophosphatase (EC 3.6.1.1) revealed a regulatory PPi-binding site. Rate vs substrate concentration dependencies were markedly nonhyperbolic in the range of 0.1-150 microM MgPPi at fixed Mg2+ levels of 0.05-10 mM provided that the enzyme had been preequilibrated with Mg2+. Imidodiphosphate, hydroxymethylenebisphosphonate, and phosphate eliminated the deviations from the Michaelis-Menten kinetics and inhibited PPi hydrolysis in a manner consistent with their binding at both active and regulatory sites. The results agreed with a model in which binding of uncomplexed PPi at the regulatory site markedly increases enzyme affinity for the activating Mg2+ ion. Ultrafiltration studies revealed the binding of at least 3 mol of the inhibitory hydroxymethylenebisphosphonate and of 2 mol of noninhibitory methylenebisphosphonate per mole of the dimeric enzyme.