Biomaterial Database

The kinetic mechanism of yeast inorganic pyrophosphatase. 1987

R J Barry, and D Dunaway-Mariano

Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008274	Magnesium	A metallic element that has the atomic symbol Mg, atomic number 12, and atomic weight 24.31. It is important for the activity of many enzymes, especially those involved in OXIDATIVE PHOSPHORYLATION.
D010761	Phosphorus Radioisotopes	Unstable isotopes of phosphorus that decay or disintegrate emitting radiation. P atoms with atomic weights 28-34 except 31 are radioactive phosphorus isotopes.	Radioisotopes, Phosphorus
D011755	Pyrophosphatases	A group of enzymes within the class EC 3.6.1.- that catalyze the hydrolysis of diphosphate bonds, chiefly in nucleoside di- and triphosphates. They may liberate either a mono- or diphosphate. EC 3.6.1.-.	Pyrophosphatase
D004789	Enzyme Activation	Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.	Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D012441	Saccharomyces cerevisiae	A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement.	Baker's Yeast,Brewer's Yeast,Candida robusta,S. cerevisiae,Saccharomyces capensis,Saccharomyces italicus,Saccharomyces oviformis,Saccharomyces uvarum var. melibiosus,Yeast, Baker's,Yeast, Brewer's,Baker Yeast,S cerevisiae,Baker's Yeasts,Yeast, Baker
D015032	Zinc	A metallic element of atomic number 30 and atomic weight 65.38. It is a necessary trace element in the diet, forming an essential part of many enzymes, and playing an important role in protein synthesis and in cell division. Zinc deficiency is associated with ANEMIA, short stature, HYPOGONADISM, impaired WOUND HEALING, and geophagia. It is known by the symbol Zn.
D043564	Inorganic Pyrophosphatase	An enzyme which catalyzes the hydrolysis of diphosphate (DIPHOSPHATES) into inorganic phosphate. The hydrolysis of pyrophosphate is coupled to the transport of HYDROGEN IONS across a membrane.	Pyrophosphatase, Inorganic,H(+)-PPase,H+-Pyrophosphatase,Proton-Pumping Inorganic Pyrophosphatase,Proton-Translocating Pyrophosphatase,Pyrophosphate-Energized Inorganic Pyrophosphatase,H+ Pyrophosphatase,Inorganic Pyrophosphatase, Proton-Pumping,Inorganic Pyrophosphatase, Pyrophosphate-Energized,Proton Pumping Inorganic Pyrophosphatase,Proton Translocating Pyrophosphatase,Pyrophosphatase, Proton-Pumping Inorganic,Pyrophosphatase, Proton-Translocating,Pyrophosphatase, Pyrophosphate-Energized Inorganic,Pyrophosphate Energized Inorganic Pyrophosphatase