The pyrophosphatase activity of pig kidney alkaline phosphatase and its inhibition by magnesium ions and excess of pyrophosphate

1. Pig kidney enzyme resembles other non-specific alkaline phosphatases in its ability to hydrolyse inorganic pyrophosphate (PP(i)). 2. Studies of enzyme velocity as a function of PP(i) concentration show that Michaelis-Menten kinetics are obeyed when a constant PP(i)/Mg(2+) concentration ratio is maintained, but velocity-substrate concentration curves are sigmoid when the concentration of PP(i) is increased but that of Mg(2+) is kept constant. The enzyme is inhibited when the total PP(i) concentration is greater than the total concentration of Mg(2+). Pyrophosphatase activity is activated by Mg(2+), but if the concentration of the metal ion is increased to a value in excess of the total PP(i) concentration Mg(2+) is then strongly inhibitory. 4. It appears that the enzyme is most active towards the complex ion MgPP(i) (2-). The enzyme probably hydrolyses PP(i) (4-) also, but this is a poorer substrate and its competition with MgPP(i) (2-) leads to inhibition. At high Mg(2+) concentrations Mg(2)PP(i) is formed. This complex appears to be a potent inhibitor. 5. Sigmoid plots of v against s and of v against i result from interactions occurring between Mg(2+) and PP(i) (4-) leading to MgPP(i) (2-) and Mg(2)PP(i), and are not indicative of allosteric behaviour.     

The distribution of l-asparagine synthetase in the principal organs of several mammalian and avian species

1. Sulphate-dependent PP(i)-ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP(2-) and MgP(2)O(7) (2-); ATP sulphurylase activity was not correlated with the concentration of free Mg(2+). 2. Sulphate-dependent PP(i)-ATP exchange was independent of PP(i) concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PP(i)-ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [(32)P]ATP from [(32)P]PP(i) and adenosine 5'-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PP(i) and adenosine 5'-sulphatophosphate. 5. The synthesis of ATP from PP(i) and adenosine 5'-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PP(i) and adenosine 5'-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5'-sulphatophosphate and non-competitive with respect to PP(i). It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP(2-) was the first product to react with the enzyme and MgP(2)O(7) (2-) was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5'-sulphatophosphate could not be demonstrated.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

Adenine nucleotides and magnesium ions in relation to control of mammalian cerebral-cortex hexokinase

1. The kinetics of inhibition of brain soluble cytoplasmic hexokinase by ADP were examined in relation to variations in the concentrations of Mg(2+) and ATP. The type of inhibition observed was dependent on the Mg(2+)/ATP ratio. 2. ADP at Mg(2+)/ATP ratios 2:1 exhibited inhibition of the ;mixed' type; at Mg(2+)/ATP ratios 1:1 the inhibition appeared to be competitive with regard to ATP. 3. Inhibition by free ATP was observed when the Mg(2+)/ATP ratio was less than 1:1. The inhibition was also of the ;mixed' type with respect to MgATP(2-). 4. The inhibitions due to ADP and to free ATP were not additive. The results suggested that there may be up to four sites in the soluble enzyme: for glucose, glucose 6-phosphate, ADP and MgATP(2-). 5. The ;free' non-particulate intracellular Mg(2+) concentration was measured and concluded to be about 1.5mm. 6. The concentrations in vivo of Mg(2+) and ATP likely to be accessible to a cytoplasmic enzyme are suggested to be below those that yield maximum hexokinase rates in vitro. The enzymic rates were measured at relevant suboptimum concentrations of Mg(2+) and ATP in the presence of ADP. Calculations that included non-competitive inhibition due to glucose 6-phosphate (56-65% at 0.25mm) resulted in net rates very similar to the measured rates for overall glycolysis. This system may therefore provide a basis for effective control of cerebral hexokinase.     

Factors affecting the activity of pyruvate kinase of Acetobacter xylinum

1. Extracts of Acetobacter xylinum were found to contain the glycolytic enzymes involved in the conversion of triose phosphate into pyruvate. Pyruvate kinase had the lowest relative activity. Phosphofructokinase activity was not detected in the extracts. 2. Only slight differences in the activity of pyruvate kinase were observed between cells grown on glucose and those grown on intermediates of the tricarboxylic acid cycle. 3. Pyruvate kinase, partially purified from ultrasonic extracts by ammonium sulphate fractionation, required Mg(2+) ions for activity. It was not activated by K(+) or NH(4) (+) ions. 4. The plots representing the relationship between initial velocity and phosphoenolpyruvate concentration were sigmoidal, suggesting a co-operative effect for phosphoenolpyruvate. The Hill coefficient (n) for phosphoenolpyruvate was 2. The rate of the reaction changed with increasing ADP concentrations according to normal Michaelis-Menten kinetics. 5. The enzyme was inhibited by ATP (K(i)0.9x10(-3)m). The inhibition was competitive with regard to ADP but not with regard to phosphoenolpyruvate. It was not relieved by excess of Mg(2+) ions. 6. The possible relationship of the properties of pyruvate kinase to regulatory mechanisms for controlling gluconeogenesis and carbohydrate oxidation in A. xylinum is discussed.     

Magnesium dependence of the measured equilibrium constants of aminoacyl-tRNA synthetases

The apparent equilibrium constants (K') for six reactions catalyzed by aminoacyl-tRNA synthetases from Escherichia coli were measured, the equations for the magnesium dependence of the equilibrium constants were derived, and best-fit analyses between the measured and calculated values were used. The K' values at 1 mM Mg(2+) ranged from 0.49 to 1.13. The apparent equilibrium constants increased with increasing Mg(2+) concentrations. The values were 2-3 times higher at 20 mM Mg(2+) than at 1 mM Mg(2+), and the dependence was similar in the class I and class II synthetases. The main reason for the Mg(2+) dependence is the existence of PP(i) as two magnesium complexes, but only one of them is the real product. AMP exists either as free AMP or as MgAMP, and therefore also has some effect on the measured equilibrium constant. However, these dependences alone cannot explain the measured results. The measured dependence of the K' on the Mg(2+) concentration is weaker than that caused by PP(i) and AMP. Different bindings of the Mg(2+) ions to the substrate tRNA and product aminoacyl-tRNA can explain this observation. The best-fit analysis suggests that tRNA reacts as a magnesium complex in the forward aminoacylation direction but this given Mg(2+) ion is not bound to aminoacyl-tRNA at the start of the reverse reaction. Thus Mg(2+) ions seem to have an active catalytic role, not only in the activation of the amino acid, but in the posttransfer steps of the aminoacyl-tRNA synthetase reaction, too.     

The enzymology of short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata. Kinetic studies and a proposed reaction mechanism

1. Short-chain fatty acyl-CoA synthetase from seeds of Pinus radiata was examined by acetate- and propionate-dependent PP(i)-ATP exchange. Reaction mixtures came to equilibrium almost instantly as judged by rates of exchange and analysis of an incubation mixture. 2. The activity of the enzyme was correlated with the concentration of MgP(2)O(7) (2-) but not with the concentration of Mg(2+), as judged by PP(i)-ATP exchange and fatty acyl AMP-dependent synthesis of ATP in the presence of PP(i). In PP(i)-ATP exchange assays, no clear relationship between activity and any single species of ATP was apparent. 3. High concentrations of fatty acid inhibited PP(i)-ATP exchange. PP(i)-dATP exchange was less than PP(i)-ATP exchange at low concentrations of fatty acid, but at higher concentrations PP(i)-dATP exchange exceeded PP(i)-ATP exchange. The rate of synthesis of fatty acyl-CoA in the presence of dATP was less than with ATP. 4. ATP and propionate inhibited the synthesis of ATP from propionyl-AMP and PP(i). The inhibition by ATP was competitive with respect to propionyl-AMP and non-competitive with respect to PP(i). The inhibition by propionate was non-competitive with respect to propionyl-AMP and PP(i). 5. AMP was a competitive inhibitor of propionyl-AMP-dependent synthesis of ATP and competitively inhibited propionate-dependent PP(i)-ATP exchange when ATP was the variable substrate. 6. It was concluded that the first partial reaction catalysed by the enzyme is ordered; ATP is the first substrate to react with the enzyme and PP(i) is probably the only product released.     

Some aspects of the kinetics of rat liver pyruvate carboxylase

1. The kinetics of rat liver pyruvate carboxylase were examined and the effect of various agents as activators or inhibitors determined. 2. Essentially similar results were obtained in comparisons of kinetics determined by a radioactivity method involving extracts of acetone-dried powders from whole livers and with a spectrophotometric assay using partially purified enzyme from the mitochondrial fraction. Activity per g of liver from fed or starved rats assayed under optimum substrate and activator conditions was 3 or 6 mumol of oxaloacetate formed/min at 30 degrees C, respectively. 3. The enzyme exhibited cold-lability and lost activity on standing, even in 1.5m-sucrose. 4. The K(m) towards pyruvate was about 0.33mm and towards bicarbonate 4.2mm. K(m) towards MgATP(2-) was 0.14mm. Mg(2+) ions activated the enzyme, in addition to their role in MgATP(2-) formation. From calculations of likely concentrations of free Mg(2+) in the assay medium a K(a) towards Mg(2+) of about 0.25mm was deduced. Mn(2+) also activated the enzyme as well as Mg(2+), but at much lower concentrations. It appeared to be inhibitory when concentrations of free Mn(2+) as low as 0.1mm were present. 5. Excess of ATP is inhibitory, and this appears at least in part independent of the trapping of Mg(2+). 6. Both Co(2+) and Zn(2+) were inhibitory; 2mol of the latter appeared to be bound even in the presence of excess of Mg(2+) and the inhibition was time-dependent. 7. Ca(2+) inhibited by competition with Mg(2+) (K(i) about 0.38mm). The inhibition due to Ca(2+) was less pronounced when activation was with Mn(2+). Inhibition by Ca(2+) and ATP appeared to be additive. 8. Hill plots suggested that no interactions occurred between ATP-binding sites. Although similar plots for total Mg(2+) gave n=3.6, no conclusions could be drawn due to the chelation of the cation with other components of the assay. Similar difficulties arose in assessing the values for Ca(2+). 9. The enzyme was inactive in the absence of acetyl-CoA and showed a sigmoidal response in its presence. K(a) was about 0.1mm with possibly up to four binding sites. Malonyl-CoA was a competitive inhibitor, with K(i) 0.01mm. 10. There was no apparent inhibition by glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, acetoacetate, beta-hydroxybutyrate, malate, aspartate, pyruvate, palmitoylcarnitine, octanoate, glutathione, butacaine, triethyltin or potassium chloride under the conditions used. Inhibition was found with citrate (possibly by chelation) and adenosine, and also by phosphoenolpyruvate, AMP, ADP and cyclic AMP, K(i) towards the last four being 0.55, 0.76, 0.25 and 1.4mm respectively.     

Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow-trout fructose diphosphatase

1. The properties of fructose diphosphatase from the liver of rainbow trout (Salmo gairdnerii) were examined over the physiological temperature range of the organism. 2. Saturation curves for substrate (fructose 1,6-diphosphate) and a cofactor (Mg(2+)) are sigmoidal, and Hill plots of the results suggest a minimum of two interacting fructose 1,6-diphosphate sites and two interacting Mg(2+) sites per molecule of enzyme. 3. Mn(2+)-saturation curves are hyperbolic, and the K(a) for Mn(2+), which inhibits the enzyme at high concentrations, is 50-100-fold lower than the K(a) for Mg(2+). 4. Fructose diphosphatase is inhibited by low concentrations of AMP; this inhibition appears to be decreased and reversed by increasing the concentrations of Mg(2+) and Mn(2+). Higher concentrations of AMP are required to inhibit the trout fructose diphosphatase in the presence of Mn(2+). 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be temperature-independent, whereas the affinities for Mg(2+) and AMP are highly temperature-dependent. 6. The pH optimum of the enzyme depends on the concentrations of Mg(2+) and Mn(2+). In addition, pH determines the K(a) for Mg(2+); at high pH, K(a) for Mg(2+) is lowered. 7. The enzyme is inhibited by Ca(2+) and Zn(2+), and the inhibition is competitive with respect to both cations. 8. The possible roles of these ions and AMP in the modulation of fructose diphosphatase and gluconeogenic activity are discussed in relation to temperature adaptation.     

Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.     

Polyvalent cation-sensing mechanism increased Na(+)-independent Mg(2+) transport in renal epithelial cells

Extracellular Ca(2+)/polyvalent cation-sensing receptor (CaSR) is capable of monitoring changes in extracellular polyvalent cation concentrations. In the present study, we investigated whether CaSR agonists reinforce the decrease of intracellular free Mg(2+) concentration ([Mg(2+)](i)) induced by extracellular Mg(2+) plus Na(+) removal. Interestingly, exposure of NRK-52E renal epithelial cells to increasing extracellular Mg(2+) concentrations from 0.8 to 15 mM for 1-2 days resulted in a twofold increase in the levels of CaSR mRNA and protein. By fluorophotometer (with mag-fura 2 fluorescent dye) and atomic absorption spectrophotometer, we confirmed that activation of CaSR by neomycin (0.5 mM) or gadolinium (1 mM) reinforced the decrease of [Mg(2+)](i) induced by Mg(2+) removal in the cells cultured in 10 mM Mg(2+)-containing medium. The neomycin-induced [Mg(2+)](i) decrease was inhibited by nicardipine (50 microM), but not by verapamil (50 microM) or amiloride (0.1 mM). These results indicate that CaSR monitors extracellular Mg(2+) concentration, and probably cause activation of Na(+)-independent Mg(2+)-transport system.     

Steady-state kinetic properties of phosphoglycerate kinase of mung beans

Steady-state kinetics of the action of mung bean phosphoglycerate kinase have been investigated using 3-phosphoglycerate and ATP as substrates in the presence of Mg2+ ions. Keeping a constant and high Mg2+ concentration and varying the concentration of one of the substrates (ATP or 3-phosphoglycerates) at several fixed concentrations of the other substrate (3-phosphoglycerate or ATP), the Km values of Mg.ATP2- and 3-phosphoglycerate were found to be 0.42 and 0.60 mM, respectively. These values are independent of the concentration of the other substrate. A limiting value of Vmax, where the enzyme is saturated with both the substrates, was found to be 39.4 mumoles product formed per min per mg enzyme protein. This corresponds to a turnover number equal to 31.5 sec-1 (for molecular weight of the enzyme equal to 48,000). If [Mg2+] and [ATP4-] are held equal and varied together at several fixed concentrations of 3-phosphoglycerate, deviations from Michaelis-Menten kinetics (non-linear Lineweaver-Burk plots) are observed at lower values of ATP4- and Mg2+ (less than 0.1 mM), giving rise to apparent sigmoidicity in the rate versus [ATP4-] plots. It has been suggested that the real substrate for this enzyme is the Mg.ATP2- complex (and not free ATP4-). The complex dissociates at lower values of [Mg2+] and [ATP4-]. The resulting disproportionate decrease in the concentration of the complex brings about a steeper fall in the rate of reaction than is required by the Michaelis-Menten equation, giving rise to an apparent sigmoidicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanylyl cyclase-activating proteins (GCAPs) are Ca2+/Mg2+ sensors: implications for photoreceptor guanylyl cyclase (RetGC) regulation in mammalian photoreceptors

Guanylyl cyclase-activating proteins (GCAP) are EF-hand Ca(2+)-binding proteins that activate photoreceptor guanylyl cyclase (RetGC) in the absence of Ca(2+) and inhibit RetGC in a Ca(2+)-sensitive manner. The reported data for the RetGC inhibition by Ca(2+)/GCAPs in vitro are in disagreement with the free Ca(2+) levels found in mammalian photoreceptors (Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., and Fain, G. L. (2002) J. Physiol. (Lond.) 542, 843-854). We have found that binding of Mg(2+) dramatically affects both Ca(2+)-dependent conformational changes in GCAP-1 and Ca(2+) sensitivity of RetGC regulation by GCAP-1 and GCAP-2. Lowering free Mg(2+) concentrations ([Mg](f)) from 5.0 mm to 0.5 mm decreases the free Ca(2+) concentration required for half-maximal inhibition of RetGC ([Ca]((1/2))) by recombinant GCAP-1 and GCAP-2 from 1.3 and 0.2 microm to 0.16 and 0.03 microm, respectively. A similar effect of Mg(2+) on Ca(2+) sensitivity of RetGC by endogenous GCAPs was observed in mouse retina. Analysis of the [Ca]((1/2)) changes as a function of [Mg](f) in mouse retina shows that the [Ca]((1/2)) becomes consistent with the range of 23-250 nm free Ca(2+) found in mouse photoreceptors only if the [Mg](f) in the photoreceptors is near 1 mm. Our data demonstrate that GCAPs are Ca(2+)/Mg(2+) sensor proteins. While Ca(2+) binding is essential for cyclase activation and inhibition, Mg(2+) binding to GCAPs is critical for setting the actual dynamic range of RetGC regulation by GCAPs at physiological levels of free Ca(2+).     

The inhibition of skeletal-muscle fructose 1,6-diphosphatase by adenosine monophosphate

1. The present study extends the finding of Krebs & Woodford (1965) that muscle fructose diphosphatase is more sensitive to AMP inhibition than liver fructose diphosphatase. 2. Hen breast fructose diphosphatase has a K(i) for AMP of 0.1mum; the plot of percentage inhibition is non-sigmoid and the reciprocal plot of activity against AMP concentration is sometimes linear. 3. Percentage inhibition plots for other muscle fructose diphosphatases are sigmoid curves which exhibit different threshold responses to the AMP concentration. 4. The intracellular content of AMP in all muscles tested exceeds the inhibition concentration range of AMP. 5. The sensitivity of muscle fructose diphosphatase to AMP inhibition is decreased by the presence of Mg(2+) or Mn(2+) ions; in the presence of Mn(2+) the inhibition curve for hen breast fructose diphosphatase becomes sigmoid. 6. From the formation constants for the Mg(2+) and Mn(2+) chelates, the effect of these ions in chelation of AMP can be calculated. Although chelation of AMP can explain the Mg(2+) effect, it cannot explain the marked relief of AMP inhibition by Mn(2+). 7. It is suggested that Mn(2+) has a specific effect on this enzyme which reduces the sensitivity to AMP inhibition.     

Studies of Mg2+/Ca2+ complexes of naturally occurring dinucleotides: potentiometric titrations, NMR, and molecular dynamics

Dinucleotides (Np(n)N'; N and N' are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg(2+) and Ca(2+) complexes of Np(n)N's. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap(n)A-Mg(2+), n = 3, 4, 5, respectively). The base (A or G) or ion (Mg(2+) or Ca(2+)) has a minor effect on K(M)(ML) values. In a physiological medium, the longer Ap(n)As (n = 4, 5) are predicted to occur mostly as the Mg(2+)/Ca(2+) complexes. (31)P NMR monitored titrations of Np(n)N's with Mg(2+)/Ca(2+) ions showed that the middle phosphates of the dinucleotides coordinate with Mg(2+)/Ca(2+). Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap(2)A and Ap(4)A coordinate Mg(2+) and Ca(2+) ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np(n)N'-Mg(2+)/Ca(2+) complexes remains the same as that of free mononucleotides.     

Probing essential water in yeast pyrophosphatase by directed mutagenesis and fluoride inhibition measurements

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.     

Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H(2)O(2)) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca(2+)/Mg(2+)-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca(2+)/Mg(2+)-supplemented medium. In Ca(2+)/Mg(2+)-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 microm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H(2)O(2) (84.40+/-0.50 ng/egg) when compared to control eggs (80.46+/-1.34 ng/egg). The higher concentration of calcium ionophore (1.6 microm) induced apoptosis and pronounced generation of intracellular H(2)O(2) (92.43+/-0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H(2)O(2) level (81.20+/-1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H(2)O(2) in rat eggs.     

Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism

Hepatitis delta virus (HDV) uses genomic and antigenomic ribozymes in its replication cycle. We examined ribozyme self-cleavage over eight orders of magnitude of Mg(2+) concentration, from approximately 10(-9) to 10(-1) M. These experiments were carried out in 1 M NaCl to aid folding of the ribozyme and to control the ionic strength. The concentration of free Mg(2+) ions was established using an EDTA-Mg(2+) buffered system. Over the pH range of 5-9, the rate was independent of Mg(2+) concentration up to 10(-7) M, and of the addition of a large excess of EDTA. This suggests that in the presence of 1 M NaCl, the ribozyme can fold and cleave without using divalent metal ions. Br?nsted analysis under these reaction conditions suggests that solvent and hydroxide ions may play important roles as general base and specific base catalysts. The observed rate constant displayed a log-linear dependence on intermediate Mg(2+) concentration from approximately 10(-7) to 10(-4) M. These data combined with the shape of the pH profile under these conditions are consistent with the binding of at least one structural divalent metal ion that does not participate in catalysis and binds tighter at lower pH. No evidence for a catalytic role for Mg(2+) was found at low or intermediate Mg(2+) concentrations. Addition of Mg(2+) to physiological and higher concentrations, from 10(-3) to 10(-1) M, revealed a second saturable divalent metal ion which binds tighter at high pH. The shape of the pH profile is inverted relative to that at low Mg(2+) concentrations, consistent with a general acid-base catalysis mechanism in which a cytosine (C75) acts as the general acid and a hydroxide ion from the divalent metal ion, or possibly from solvent, acts as the base. Overall, the data support a model in which the HDV ribozyme can self-cleave by multiple divalent ion-independent and -dependent channels, and in which the contribution of Mg(2+) to catalysis is modest at approximately 25-fold. Surface electrostatic potential maps were calculated on the self-cleaved form of the ribozyme using the nonlinear Poisson-Boltzmann equation. These calculations revealed several patches of high negative potential, one of which is present in a cleft near N4 of C75. These calculations suggest that distinct catalytic and structural metal ion sites exist on the ribozyme, and that the negative potential at the active site may help shift the pK(a) for N3 of C75 toward neutrality.     

Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase

1. The properties of fructose diphosphatase from liver of South American lungfish (Lepidosiren paradoxa) were examined. 2. Saturation curves for substrate (fructose diphosphate) and both cofactors (Mn(2+) and Mg(2+)) are sigmoidal and Hill plots of these results suggest about 2 interacting substrate and cofactor sites/molecule of enzyme. 3. Mn(2+) is an efficient positive modulator of the enzyme and K(a) for Mn(2+) is about 20-30-fold lower than the K(a) for Mg(2+). 4. Lungfish fructose diphosphatase is inhibited by low concentrations of AMP, and the affinity of the enzyme for AMP is insensitive to temperature. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be dependent on temperature, whereas affinity for Mg(2+) is temperature-independent. 6. The pH optimum of the enzyme depends on the presence of the particular cofactor. As pH increases, the K(a) values of both cations are lowered, maximum velocities are increased and the saturation curves for cofactor become hyperbolic. 7. The possible roles of these ions, pH and substrate in the modulation of fructose diphosphatase and gluconeogenic activity in the lungfish are discussed in relation to aestivation and temperature adaptation.