The roles of magnesium ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The involvement of Mg2+ ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei was studied. The true substrate for the enzyme was shown to be the MgATP2-complex, and free Mg2+ ions are also required for enzyme activity. At concentrations of MgATP2- of 2.92 mM and greater, and a fructose 6-phosphate concentration of 1 mM and in the presence of EDTA as a Mg2+ buffer, the Km value for Mg2+ was determined to be 294 +/- 18 microM. Neither MgATP nor free ATP is an inhibitor of the enzyme, although apparent inhibition by the latter can be observed as a consequence of the decrease in free Mg2+ by chelation.     

Comparative enzymatic properties of avian liver and skeletal muscle D-fructose-1,6-bisphosphate 1-phosphohydrolase.

The enzymatic properties of purified preparations of chicken liver and chicken skeletal muscle fructose bisphosphatases (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) were compared. Both enzymes have an absolute requirement for Mg2+ or Mn2+. The apparent Km for MgCl2 at pH 7.5 was 0.5 mM for the muscle enzyme and 5 mM for the liver enzyme. Fructose bisphosphate inhibited both enzymes. At pH 7.5, the inhibitor constants (Ki) were 0.18 and 1.3 mM for muscle and liver fructose bisphosphatases, respectively. The muscle enzyme was considerably more sensitive to AMP inhibition than the liver enzyme. At pH 7.5 and in the presence of 1 mM MgCl2, 50% inhibition of muscle and liver fructose bisphosphatases occurred at AMP concentrations of 7 X 10(-9) and 1 X 10(-6) M, respectively. EDTA activated both enzymes. The degree of activation was time and concentration dependent. The degree of EDTA activation of both enzymes decreased with increasing MgCl2 concentration. Ca2+ was a potent inhibitor of both liver (Ki, 1 X 10(-4) M) and muscle (Ki, 1 X 10(-5) M) fructose bisphosphatase. This inhibition was reversed by the presence of EDTA. Ca2+ appears to be a competitive inhibitor with regard to Mg2+. There is, however, a positive homeotropic interaction among Mg2+ sites of both enzymes in the presence of Ca2+.     

Control of rabbit liver fructose-1, 6-diphosphatase activity by magnesium ions.

EDTA at a concentration of 1 muM produced a threshold effect in the activation of purified rabbit liver fructose-1, 6-diphosphatase [EC 3.1.3.11] in the presence of 5 mM Mg2+ at pH 7.2. Without EDTA, biphasic activation curves were produced by Mg2+. A double-reciprocal plot of the data gave the Km values corresponding to the two linear regions. They were 0.19 and 0.83 mM at pH 7.5, and 0.055 and 0.83 mM at pH 9.1. In the presence of 5muM EDTA a sigmoidal curve was obtained for Mg2+ activation in the range of noninhibitory Mg2+ concentrations at pH 7.2. The apparent Km value for Mg2+ was 0.15 mM, and the Hill coefficient was 2.0. At pH 9.1 cooperativity among the Mg2+ sites disappeared, and the apparent Km value for Mg2+ was 0.055 mM. These Km values at pH 7.2 or 9.1 corresponded to the smaller of the biphasic Km values obtained without EDTA. In the absence of EDTA, no inhibition by Mg2+ was observed in the Mg2+ concentration range below 10 mM. In the presence of EDTA, the enzyme was inhibited markedly by Mg2+ at concentrations above 0.5 mM at pH 7.2, and was more sensitive to inhibition at pH 9.1. The effects of pH on the Km value for Mg2+ activation and on the Mg2+ inhibition contributed to an apparent shift of the pH optimum for activity induced by EDTA. Cooperative interaction among fructose-1, 6-diphosphate sites was observed for the enzyme in the presence of EDTA. The Hill coefficient was approximatley 1.8, and the apparent Km value for the substrate was 0.74 muM. EDTA appears to make liver fructose-1, 6-diphosphatase very sensitive to various effectors. It is suggested that Mg2+ serves as a regulator for the enzyme activity.     

Kinetic studies on the regulation of rabbit liver pyruvate kinase

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K(+) and optimum activity was recorded with 30mm-K(+), 4mm-MgADP(-), with a MgADP(-)/ADP(2-) ratio of 50:1, but inhibition occurred with K(+) concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg(2+) was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (n(H)=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent K(m) for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis-Menten response was obtained when phosphoenolpyruvate was the variable substrate (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).     

Purification and properties of spinach leaf cytoplasmic fructose-1,6-bisphosphatase.

Cytoplasmic fructose-1,6-bisphosphatase has been purified from spinach leaves to apparent homogeneity. The enzyme is a tetramer of molecular weight about 130,000. At pH 7.5, the Km for fructose 1.6-bisphosphate was 2.5 micron, and for MgCl2 0.13 mM; the enzyme was specific for fructose 1,6-bisphosphate. Saturation with Mg2+ was achieved with lower concentrations at pH 8 than at pH 7. AMP and high concentrations of fructose 1,6-bisphosphate inhibited enzyme activity. Ammonium sulfate relieved the latter inhibition but was itself inhibitory when substrate concentrations were low. Acetylation studies demonstrated that the AMP regulatory site was distinct from the catalytic site. Cytoplasmic fructose-1,6-bisphosphatase may contribute to the regulation of sucrose biosynthesis in plant leaves.     

Inhibition of inorganic pyrophosphatase of animal mitochondria by calcium

Calcium ion is an uncompetitive inhibitor of the inorganic pyrophosphatases of bovine heart and rat liver mitochondria with respect to substrate MgPPi at pH 8.5 and a non-competitive inhibitor of the former enzyme at pH 7.2. The concentration of Ca2+ required to decrease the maximal velocities for both enzymes at pH 8.5, 0.4 mM Mg2+ was about 10 microM. The inhibition results from the binding of two Ca2+ ions to both free enzymes and their complexes with the substrate. The results suggest that Ca2+ regulates pyrophosphatase activity and hence PPi level in mammalian mitochondria.     

Arginine decarboxylase from a Pseudomonas species.

An arginine decarboxylase has been isolated from a Pseudomonas species. The enzyme is constitutive and did not appear to be repressed by a variety of carbon sources. After an approximately 40-fold purification, the enzyme appeared more similar in its properties to the Escherichia coli biosynthetic arginine decarboxylase than to the E. coli inducible (biodegradative) enzyme. The Pseudomonas arginine decarboxylase exhibited a pH optimum of 8.1 and an absolute requirement of Mg2+ and pyridoxal phosphate, and was inhibited significantly at lower Mg2+ concentrations by the polyamines putrescine, spermidine, and cadaverine. The Km for L-arginine was about 0.25 mM at pH 8.1 AND 7.2. The enzyme was completely inhibited by p-chloromercuribenzoate. The inhibition was prevented by dithiothreitol, a feature that suggests the involvement of an -SH group. Of a variety of labeled amino acids tested, only L-arginine, but not D-arginine was decarboxylated. D-Arginine was a potent inhibitor of arginine decarboxylase with a Ki of 3.2 muM.     

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.     

The allosteric properties of beef-liver fructose bisphosphatase.

1. The activity of beef liver fructose bisphosphatase has been shown to respond cooperatively to increasing concentrations of the activating cations Mg2+ and Mn2+. The allosteric inhibitor AMP caused an increase in this cooperativity and a decrease in the apparent affinity of the enzyme for the activating cation. 2. The cooperative response of the enzyme to AMP is similarly increased by increasing cation concentrations with a concomitant decrease in the apparent affinity. 3. Direct binding experiments indicated that in the absence of either Mg2+ or Mn2+ the enzyme bound AMP non-cooperatively up to a maximum of two molecules per molecule of enzyme, a result that is indicative of half-sites reactivity. The binding became increasingly cooperative as the concentration of the activating cation was increased. 4. The substrate fructose bisphosphate had no effect on any of these cooperative responses. 5. These results may be most simply interpreted in terms of concerted model in which the activating cation functions both as an allosteric activator and as an essential cofactor for the reaction.     

Carbohydrate structures of the third component of rat complement. Presence of both high-mannose and complex type oligosaccharide chains.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the concentration of the effector fructose 1,6-bisphosphate. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that an increase in the fructose bisphosphate concentration from 24 microM to 1.2 mM brings about a transition from a sigmoidal to a non-inflected form in the relationships v = f([phosphoenolpyruvate]) and v = f([Mg2+]) together with a large increase in the affinity of these substrates for the enzyme. The binding behaviour of ADP is barely affected by the same change in effector concentration. By contrast, increase in fructose bisphosphate concentration below 24 microM increases the affinity of the enzyme for all its substrates and the sigmoidicity of the corresponding velocity-substrate-concentration relationships. As a result of this change in behaviour it has been found impossible to represent all the data by the exponential model for a regulatory enzyme, and it is suggested (supported by comparisons with previous work) that the failure may reflect a secondary action of the effector upon the enzyme.     

Kinetic properties and related changes of molecular weight in a fructokinase from Streptomyces violaceoruber.

1. A study of the initial reaction rates at variable substrate concentrations and of the molecular weight of the enzyme in the presence of different effectors, has been carried out using fructokinase (ATP: fructose 6-phosphotransferase, EC 2.7.1.4) from Streptomyces violaceoruber. 2. Saturation curves for MgATP or CoATP are sigmoidal and they change to hyperbolic in the presence of 10 mM Mg2+ or Co2+ in excess over the nucleoside triphosphate. 3. Saturation cuvves for fructose show intermediary plateaux at high (but not at low) concentrations of ATP or Mg2+. 4. The molecular weight of the enzyme in the presence of high concentrations of MgATP is 80 000. In the presence of fructose, and/or Mg2+, the molecular weight is 20 000. 5. The effects of MgADP, uncomplexed ADP or ATP, and low concentrations of detergent on the kinetics have been studied. The results are interpreted as showing the existence of cooperative effects.     

Catalytic properties of alkaline phosphatase from pig kidney

The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (V(max.)) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme-substrate complex is 8.7 for p-nitrophenyl phosphate and beta-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme-substrate complex, 8.7, is very similar to that for the enzyme-substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (DeltaH) at pH9.5 showed a transition at 24.8 degrees C that was unaffected by Mg(2+). 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn(2+) ions/tetrameric enzyme for an ordered association of the monomers. Zn(2+) in excess and other bivalent ions compete for a second site with Mg(2+). Mg(2+) enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.     

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.     

Fructose bisphosphatase from Escherichia coli. Purification and characterization

Escherichia coli fructose-1,6-bisphosphatase has been purified for the first time, using a clone containing an approximately 50-fold increased amount of the enzyme. The procedure includes chromatography in phosphocellulose followed by substrate elution and gel filtration. The enzyme has a subunit molecular weight of approximately 40,000 and in nondenaturing conditions is present in several aggregated forms in which the tetramer seems to predominate at low enzyme concentrations. Fructose bisphosphatase activity is specific for fructose 1,6-bisphosphate (Km of approximately 5 microM), shows inhibition by substrate above 0.05 mM, requires Mg2+ for catalysis, and has a maximum of activity around pH 7.5. The enzyme is susceptible to strong inhibition by AMP (50% inhibition around 15 microM). Phosphoenolpyruvate is a moderate inhibitor but was able to block the inhibition by AMP and may play an important role in the regulation of fructose bisphosphatase activity in vivo. Fructose 2,6-bisphosphate did not affect the rate of reaction.     

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.     

Detection and characterization of sorbitol dehydrogenase from apple callus tissue

Sorbitol dehydrogenase (l-iditol:NAD(+) oxidoreductase, EC 1.1.1.14) has been detected and characterized from apple (Malus domestica cv. Granny Smith) mesocarp tissue cultures. The enzyme oxidized sorbitol, xylitol, l-arabitol, ribitol, and l-threitol in the presence of NAD. NADP could not replace NAD. Mannitol was slightly oxidized (8% of sorbitol). Other polyols that did not serve as substrate were galactitol, myo-inositol, d-arabitol, erythritol, and glycerol. The dehydrogenase oxidized NADH in the presence of d-fructose or l-sorbose. No detectable activity was observed with d-tagatose. NADPH could partially substitute for NADH.Maximum rate of NAD reduction in the presence of sorbitol occurred in tris(hydroxymethyl)aminomethane-HCl buffer (pH 9), or in 2-amino-2-methyl-1,3-propanediol buffer (pH 9.5). Maximum rates of NADH oxidation in the presence of fructose were observed between pH 5.7 and 7.0 with phosphate buffer. Reaction rates increased with increasing temperature up to 60 C. The K(m) for sorbitol and xylitol oxidation were 86 millimolar and 37 millimolar, respectively. The K(m) for fructose reduction was 1.5 molar.Sorbitol oxidation was completely inhibited by heavy metal ions, iodoacetate, p-chloromercuribenzoate, and cysteine. ZnSO(4) (0.25 millimolar) reversed the cysteine inhibition. It is suggested that apple sorbitol dehydrogenase contains sulfhydryl groups and requires a metal ion for full activity.     

Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Rat liver fructose-1,6-bisphosphatase, which was assayed by measuring the release of 32P from fructose 1,6-[1-32P]bisphosphate at pH 7.5, exhibited hyperbolic kinetics with regard to its substrate. beta-D-Fructose 2,6-bisphosphate, an activator of hepatic phosphofructokinase, was found to be a potent inhibitor of the enzyme. The inhibition was competitive in nature and the Ki was estimated to be 0.5 microM. The Hill coefficient for the reaction was 1.0 in the presence and absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate also enhanced inhibition of the enzyme by the allosteric inhibitor AMP. The possible role of fructose 2,6-bisphosphate in the regulation of substrate cycling at the fructose-1,6-bisphosphatase step is discussed.     

蛇肌果糖1，6－二磷酸酯酶果糖2，6－二磷酸和底物抑制的结合部位

在果糖１,６—二磷酸酯酶中果糖２,６—二磷酸可能与底物抑制的作用方式不同,因为蛇肌果糖１,６－二磷酸酯酶ｐＨ９．２的活性受到果糖２,６－二磷酸的抑制,而不受高浓度底物的影响。Ｋ＋能增强果糖２,６—二磷酸对酶活性抑制,并能较大程度地解除过量底物的抑制。快反应流基修饰酶不再受较低浓度果糖２,６—二磷酸的抑制,但高浓度果糖２,６—二磷酸仍能抑制酶活性,其ＩＣ５０增大４０倍。修饰酶受底物抑制的阈值不变。为胰蛋白酶或枯草杆菌蛋白酶限制性酶解的果糖１,６—二磷酸酯酶受过量底物和果糖２,６—二磷酸抑制的行为也不相同。以上结果可能提示在蛇肌果糖１,６—二磷酸酯酸中存在既有别于ＡＭＰ,又有别于过量底物的结合部位。     

The mechanism of rat liver fructose-1,6-bisphosphate inhibition by fructose-2,6-bisphosphate

It was found that a decrease in the activating cation (Mg2+) concentration below [A]0.5 causes the disappearance of cooperativity of the fructose 1.6-bisphosphatase substrate binding sites induced by high fructose 2.6-bisphosphate concentrations without any significant alteration in the extent of the enzyme inhibition. Under these conditions, a competitive type of inhibition (with respect to the substrate) is transformed into a non-competitive type with an increase in the fructose 2.6-bisphosphate concentration. The data obtained confirm the viewpoint that fructose 2.6-bisphosphate binds to the enzyme at two distinct sites, a catalytic and an allosteric ones, differing in their affinity for the inhibitor. It is supposed that the interaction between the allosteric fructose 2.6-bisphosphate binding site and the activator site occupied by Mg2+ is necessary for the cooperative response of the enzyme to the substrate.     

6-phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism.

The inorganic pyrophosphate-requiring 6-phosphofructokinase of Entamoeba histolytica has been further investigated. The molecular weight of the enzyme is approximately 83,000 and its isoelectric point occurs at pH 5.8 to 6.0. The divalent cation requirement for reaction was explored. In the direction of fructose 6-phosphate formation half-maximal rate required 500 muM magnesium ion; in the direction of fructose bisphosphate formation 8 muM magnesium ion sufficed. ATP, PPi, polyphosphate, acetyl phosphate, or carbamyl phosphate cannot replace PPi as phosphate donor for the conversion of fructose 6-phosphate to fructose bisphosphate. In the direction of fructose 6-phosphate formation arsenate can replace orthophosphate. Isotope exchange studies indicate that little or no exchange occurs between Pi and PPi or between fructose 6-phosphate and fructose bisphosphate in the absence of a third substrate. These findings appear to rule out phosphoenzyme formation and a ping-pong reaction mechanism. PPi, Pi, and fructose bisphosphate are competitive inhibitors of fructose bisphosphate, PPi, and fructose 6-phosphate, respectively. This argues against an ordered mechanism and suggests a random mechanism. Fructose 6-phosphate and Pi were noncompetitive with respect to each other indicating the formation of a dead end complex. These product inhibition relationships are in accord with a Random Bi Bi mechanism.