Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinities for fructose diphosphate and Mg²⁺ and were similarly activated by EDTA, dithiothreitol and cystamine.Mg²⁺, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

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.     

Binding of Zn2+ to rabbit muscle fructose 1,6-bisphosphatase and its effect on the catalytic properties.

At low concentrations (<1 μM), and in the presence of Mg²⁺, Zn²⁺ inhibits the activity of rabbit muscle fructose 1,6-bisphosphatase (EC 3.1.3.11). At higher concentrations Zn²⁺ can replace Mg²⁺ as the activating cation. The inhibitory effects of Zn²⁺ are associated with its binding to 4 high-affinity sites (1 per subunit). Binding to a second set of 4 sites requires the presence of the substrate, fructose 1,6-bisphosphate, and binding of Zn²⁺ to this set of sites restores the catalytic activity. In the absence of EDTA, Zn²⁺ is a better activating cation than Mg²⁺. The muscle enzyme differs from rabbit liver fructose 1,6-bisphosphatase in the number of binding sites (8 as compared to 12 for the rabbit liver enzyme) and in showing higher activity with Zn²⁺ as the activating cation. The results suggest that Zn²⁺ may be the physiological activator.     

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.     

Free metal ion depletion by “good's” buffers: II. N-(2-acetamido)-2-aminoethanesulfonic acid (ACESH): Complexes with Calcium(II), Magnesium(II), Manganese(II), Cobalt(II), Zinc(II), Nickel(II), and Cooper(II)

Potentiometric, visible, and infrared studies of the complexation of N-(2-acetamido)-2-aminoethanesulfonic acid (ACESH) by Ca(II), Mg(II), Mn(II), Co(II), Zn(II), Ni(II), and Cu(II) are reported. Ca(II), Mg(II), and Mn(II) were found not to complex with ACES^?, while Co(II), Zn(II), Ni(II), and Cu(II) were found to form 2:1, ACES^? to M²⁺, complexes, and [Cu(ACES)₂] was found to undergo stepwise deprotonation of the amide groups to form [Cu(H_?1ACES)₂^2?]. Formation (affinity) constants for the various metal complexes are reported, and the probable structures of the various metal chelates in solution are discussed.     

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.     

Substitution studies of the second divalent metal cation requirement of protein tyrosine kinase CSK

In addition to a magnesium ion needed to form the ATP-Mg complex, we have previously determined that at least one more free Mg2+ ion is essential for the activation of the protein tyrosine kinase, Csk [Sun, G., and Budde, R. J. A. (1997) Biochemistry 36, 2139-2146]. In this paper, we report that several divalent metal cations, such as Mn2+, Co2+, Ni2+, and Zn2+ bind to the second Mg2+-binding site of Csk with up to 13200-fold higher affinity than Mg2+. This finding enabled us to substitute the free Mg2+ at this site with Mn2+, Co2+, Ni2+, or Zn2+ while keeping ATP saturated with Mg2+ to study the role of the free metal cation in Csk catalysis. Substitution by these divalent metal cations resulted in varied levels of Csk activity, with Mn2+ even more effective than Mg2+. Co2+ and Ni2+ supports reduced levels of Csk activity compared to Mg2+. Zn2+ has the highest affinity for the second Mg2+-binding site of Csk at 0.65 microM, but supports no kinase activity, acting as a dead-end inhibitor. The inhibition by Zn2+ is reversible and competitive against free Mg2+, noncompetitive against ATP-Mg, and mixed against the phosphate accepting substrate, polyE4Y, significantly increasing the affinity for this substrate. Substitution of the free Mg2+ with Mn2+, Co2+, or Ni2+ also results in lower Km values for the peptide substrate. These results suggest that the divalent metal activator is an important element in determining the affinity between Csk and the phosphate-accepting substrate.     

Distribution and properties of a ‘lectin- like’ glycoprotein from leaves and stems of<Emphasis Type="Italic">Dolichos biflorus</Emphasis>: Implications on the role of lectins in plants

Cardiotoxin II of the Indian cobra(Naja naja) contains approximately four Mg²⁺ per mol. Complete demetallation of the toxin is achieved by three cycles of treatment with ethylenediamine tetraacetate and gel filtration. Reconstitution of toxin by treatment of the apo-protein with Mg²⁺ restores metal content and inorganic pyrophosphatase activity only to the extent of two atoms/mol and 65%, respectively. Use of Mg (II)-EDTA in the reconstitution experiment yields restoration of half the original enzyme activity. Mg²⁺ is required for the inorganic pyrophosphatase action of the toxin. A definitive statement on the non-essentiality of Mg²⁺ for the lethal toxicity of the toxin is not possible at present, although experimental observations indicate that demetallated toxin is as toxic as the native toxin. Based on this and the differing sensitivities of the enzyme and toxic activities of the toxin to heat, it is suggested that the reaction centres in the toxin for the two activities are different and that the pyrophosphatase activity is not causally connected with the lethal toxicity of the toxin     

Free metal ion depletion by "Good's" buffers. I. N-(2-acetamido)iminodiacetic acid 1:1 complexes with calcium(ii). magnesium(II), zinc(II), manganese(II), cobalt(II), nickel(II), and copper(II).

Formation (affinity) constants for 1:1 complexes of N-(2-acetamido)iminodiacetic acid (ADAH₂) with Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) have been determined. Probable structures of the various metal chelates existing in solution are discussed. Values for the deprotonation of the amide group in [Cu(ADA)] and subsequent hydroxo complex formation are also reported. The use of ADA as a buffer is considered in terms of metal buffers complexes which can be formed at physiological pH, i.e., at pH 7.0 there is essentially no free metal ion in 1:1 M²⁺ to ADA solutions.     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.     

Fructose 1,6-diphosphatase in striated muscle

1. The occurrence of fructose diphosphatase in muscle tissue was investigated with reference to the question whether lactate can be converted into glycogen in muscle, as postulated by Meyerhof (1930), fructose diphosphatase being one of the enzymes required for this conversion. 2. Fructose diphosphatase was found in skeletal muscle of man, dog, cat, rat, mouse, rabbit, guinea pig, cattle, sheep, pigeon, fowl and frog. Under the test conditions between 5 and 60 μmoles of substrate were split/g. fresh wt./hr. at 22°. 3. Like liver fructose diphosphatase, the muscle enzyme is inhibited by substrate concentrations above 0·1 mm, by AMP and by trace quantities of Zn²⁺, Fe²⁺ and Fe³⁺; it is `activated' by EDTA. Inhibitions by the above agents may account for the failure of previous authors to detect the enzyme. 4. Heart muscle of several vertebrate species and the smooth muscle of pigeon and fowl gizzard had no measurable activity. 5. The presence of fructose diphosphatase and the virtual absence of the enzyme systems converting pyruvate into phosphopyruvate means that lactate and pyruvate cannot be converted into glycogen in muscle, whereas the phosphorylated C₃ compounds can. The reconversion into carbohydrate of lactate (which readily diffuses out of muscle) occurs in liver and kidney only. The reconversion of phosphorylated C₃ intermediates (which cannot diffuse out of the tissue) can occur only within the muscle. 6. α-Glycerophosphate is probably the main intermediate requiring conversion into glycogen. The possible role of α-glycerophosphate formation in vertebrate muscle, already well established in insect muscle, is discussed.     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

Regulation in the chemolithotrophthiobacillus neapolitanus: Fructose-1,6-diphosphatase

Summary Partially purified fructose diphosphatase from the obligate chemolithotroph,Thiobacillus neapolitanus has been characterized, and some of its regulatory properties described. The enzyme had a high effinity for its substrate, but was inhibited by substrate at concentrations above 1 mM. The enzyme had an absolute requirement for a divalent cation. In the absence of EDTA there was a single pH optimum in the alkaline range between 8.5 and 9.5; in the presence of EDTA there was considerable was activity at both neutral and alkaline pH. This diphosphatase was inhibited by AMP at 10^–4 M or greater-, the lower the pH, the greater the AMP inhibition. Treatment of the enzyme with 5×10^–5 Mpara hydroxy mercuribenzoate allowed retention of full catalytic activity while abolishing considerable AMP inhibition. Exposure of the enzyme to several concentrations of urea had no effect on the AMP inhibition. Homocystine (0.06 mM) and coenzyme A (0.1 mM) had no effect. At 1 mM, PEP caused 60% inhibition, 2, 3-diphosphoglyceric acid produced 26% inhibition, and pyruvate had no effect.     

The functional significance of variants of fructose 1,6-diphosphatase in the gill and hypodermis of a marine crustacean. A kinetic study

1. In the hypodermis and gill of the Crustacea fructose 1,6-diphosphatase (EC 3.1.3.11) functions at a primary branch point between glycogen and chitin synthesis. In these tissues of the Arctic king-crab, Paralithodes camtchatica, fructose diphosphatase occurs in two electrophoretically distinguishable forms. 2. Fructose diphosphatase I (pI7.2-7.5) accounts for 70 and 10% of total fructose diphosphatase activity in the hypodermis and gill respectively, whereas fructose diphosphatase II (pI5.3) accounts for 30 and 90% of the total activity in the two tissues. Both forms display a neutral pH optimum, have an absolute requirement for a bivalent cation, and are potently inhibited by high concentrations of AMP and substrate. 3. Fructose 1,6-diphosphate saturation follows Michaelis-Menten kinetics for both fructose diphosphatases; the K(m) (fructose diphosphate) for fructose diphosphatase I is somewhat higher than for fructose diphosphatase II. In the presence of 50-200mm-K(+), the K(m) (fructose diphosphate) increases and at high concentrations of K(+) fructose diphosphate saturation follows sigmoidal kinetics. 4. UDP-N-acetylglucosamine and UDP-glucose at high concentrations specifically and potently inhibit fructose diphosphatase II, but do not significantly affect fructose diphosphatase I activity. 5. Low concentrations of UDP-N-acetylglucosamine activate fructose diphosphatase II by a decrease in the apparent K(m) (fructose diphosphate), but fructose diphosphatase I is again refractory to UDP-N-acetylglucosamine under these conditions. 6. In the presence of K(+) and UDP-N-acetylglucosamine, fructose diphosphatase II is able to compete for limiting fructose diphosphate about three times more effectively than is fructose diphosphatase I. 7. AMP inhibition of both forms of the enzyme is subject to three independent variables: (a) alkaline pH increases the K(i) (AMP), (b) K(+) decreases the K(i), increases the sigmoidicity of inhibition kinetics, increases the maximum inhibition attained, and abolishes the effect of pH on AMP inhibition, and (c) Mg(2+) strongly de-inhibits AMP-inhibited fructose diphosphatase. 8. It is postulated that the presence of two forms of fructose diphosphatase aids controlled channelling of carbon through the fructose diphosphatase ;bottleneck' either towards glycogen synthesis or chitin synthesis, but not towards both simultaneously.     

The kinetics and divalent cation inhibition of plasma membrane ATPase in the yeast Candida albicans

The kinetics of ATP hydrolysis and cation effects on ATPase activity in plasma membrane from Candida albicans ATCC 10261 yeast cells were investigated. The ATPase showed classical Michaelis-Menten kinetics for the hydrolysis of Mg X ATP, with Km = 4.8 mM Mg X ATP. Na+ and K+ stimulated the ATPase slightly (9% at 20 mM). Divalent cations in combination with ATP gave lower ATPase activity than Mg X ATP (Mg greater than Mn greater than Co greater than Zn greater than Ni greater than Ca). Divalent cations inhibited the Mg X ATPase (Zn greater than Ni greater than Co greater than Ca greater than Mn). Free Mg2+ inhibited Mg X ATPase weakly (20% inhibition at 10 mM). Computed analyses of substrate concentrations showed that free Zn2+ inhibited Zn X ATPase, mixed (Zn2+ + Mg2+) X ATPase, and Mg X ATPase activities. Zn X ATP showed high affinity for ATPase (Km = 1.0 mM Zn X ATP) but lower turnover (52%) relative to Mg X ATP. Inhibition of Mg X ATPase by (free) Zn2+ was noncompetitive, Ki = 90 microM Zn2+. The existence of a divalent cation inhibitory site on the plasma membrane Mg X ATPase is proposed.     

Regulation of divalent cations of the membrane-bound pyrophosphatase of Rhodospirillum rubrum,as shown by the hydrolysis of tripositive-pyrophosphate complexes

Tripositive-pyrophosphate [M(III)-PPi] complexes were used to investigate the role of free divalent cations on the membrane-bound pyrophosphatase. Divalent cations remain free and the M(III)-PPi complexes were employed as substrates. Formation of a La-PPi complex was studied by fluorescence, and the fact that Zn²⁺ and Mg²⁺ remain free in the solution was validated. Hydrolysis of La-PPi is stimulated by the presence of fixed concentrations of free Mg²⁺ or Zn²⁺ and this stimulation depends on the concentration of the cations when the La-PPi complex is fixed. The divalent cation stimulation order is Zn²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺ (at 0.5 mm of free cation). With different M(III)-PPi complexes, Zn²⁺ produces the same K _m, for all the complexes and Mg²⁺ stimulates with a different K _m. The results suggest that both Mg²⁺ and Zn²⁺ activate the membrane-bound pyrophosphatase but through different mechanisms.     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

Purification and properties of fructose 1,6-bisphosphatase from turkey liver.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) has been purified 360-fold from turkey liver. The purified enzyme appears to be homogeneous by disc gel electrophoresis and has a pH profile indistinguishable from that of the enzyme in crude extracts. Mn²⁺ is significantly more effective than Mg²⁺ as the essential metal cofactor of this enzyme. The maximal effect of histidine is equivalent to that of EDTA except that EDTA is more efficient at lower concentrations. The histidine effect is decreased with an increase in pH or if substrate is first bound to the enzyme. The enzyme activity is activated equally by d- and l-forms of histidine. Enzyme affinity for the substrate decreases with an increase in pH. The inhibition by high substrate concentrations observed at pH 7.5 is markedly reduced in the absence of chelating activator or when Mg² is replaced by Mn²⁺ as the metal cofactor. Turkeys liver fructose 1,6-bisphosphatase resembles the enzyme from mammalian sources in that the sensitivity to AMP inhibition is decreased with the increase in pH, temperature, and Mg² concentration.     

Effect of Mn2+ on fructose 2,6-bisphosphate inhibition of mouse liver, intestinal, and muscle fructose-1,6-bisphosphatases

Fructose 2,6-bisphosphate inhibited all three fructose-1,6-bisphosphatases from the liver, intestine, and muscle of the mouse. The sensitivity of the liver enzyme to the inhibitor was significantly diminished when Mg2+ was replaced by Mn2+ as the activating cation. Inhibition of the liver enzyme by fructose 2,6-bisphosphate decreased as the concentration of the metal activator, Mn2+ or Mg2+, increased. The respective I50 values obtained by extrapolation of metal ion concentrations to zero were 40 microM with Mn2+ and 0.25 microM with Mg2+. The extent of desensitization to either fructose 2,6-bisphosphate or AMP inhibition by Mn2+ decreased in the order of the liver, intestine, and muscle enzyme. Only in the case of the liver enzyme was the substrate cooperativity induced by fructose 2,6-bisphosphate in the presence of Mg2+. In all three isoenzymes from the mouse, fructose 2,6-bisphosphate greatly potentiated the AMP inhibition of the enzyme in the presence of either Mg2+ or Mn2+. The liver enzyme with Mn2+ in addition to Mg2+ was still active in the presence of less than 1 microM fructose 2,6-bisphosphate, even though AMP was present at 100-200 microM.