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(H/sub 2/O)4(methylenediphosphonate) (Rh(H/sub 2/O)4PCP) were used as dead-end inhibitors to study the order of binding of Cr(H/sub 2/O)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(H/sub 2/O)4PCP vs Cr(H/sub 2/O)4PP (Kis = 0.25 +/- 0.07 mM), and for RH(H/sub 2/O)4PCP vs Mg2+ (Kis = 0.38 +/- 0.03 mM). Uncompetitive inhibition was observed for Ca2+ vs Cr(H/sub 2/O)4PP (Kii = 0.49 +/- 0.01). On the basis of these results a rapid equilibrium ordered mechanism in which Cr(H/sub 2/O)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 and of Cr(H/sub 2/O)4PP. 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.
- Research Organization:
- Univ. of Maryland, College Park
- OSTI ID:
- 5719140
- Journal Information:
- Arch. Biochem. Biophys.; (United States), Vol. 259:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
COBALT COMPOUNDS
BIOLOGICAL EFFECTS
MAGNESIUM COMPOUNDS
MANGANESE COMPOUNDS
PHOSPHATASES
BIOCHEMICAL REACTION KINETICS
ZINC COMPOUNDS
CALCIUM COMPOUNDS
CATIONS
INHIBITION
PYROPHOSPHATES
SACCHAROMYCES CEREVISIAE
ALKALINE EARTH METAL COMPOUNDS
CHARGED PARTICLES
ENZYMES
ESTERASES
FUNGI
HYDROLASES
IONS
KINETICS
MICROORGANISMS
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
PLANTS
REACTION KINETICS
SACCHAROMYCES
TRANSITION ELEMENT COMPOUNDS
YEASTS
560300* - Chemicals Metabolism & Toxicology