Kinetic properties of pig pyruvate kinases type A from kidney and type M from muscle.

Kinetic properties of homogeneous preparations of pig kidney and pig muscle pyruvate kinases (EC 2.7.1.40) were studied. Both isozymes showed a hyperbolic relationship to ADP with an apparent K_m of 0.3 mm. K⁺ and Mg²⁺ were necessary for the activity of both isozymes, and their dependences on these cations were similar. The muscle isozyme expressed Michaelis-Menten type of kinetics with respect to phosphoenolpyruvate, and the apparent K_m was the same (0.03 mm) from pH 5.5 to pH 8.0. In contrast, the dependence on phosphoenolpyruvate changed with pH for the kidney isozyme. It showed similar properties to the muscle isozyme at pH 5.5–7.0 (apparent K_m of 0.08 mm), while two apparent K_m values for this substrate were present at pH 7.5–8.0, one low (0.1 mm) and one high (0.3–0.6 mm). At pH 7.5, fructose 1,6-bisphosphate converted the kidney isozyme to a kinetical form where only the lower apparent K_m for phosphoenolpyruvate was detected. On the other hand, in the presence of alanine or phenylalanine the kidney pyruvate kinase showed only the higher K_m for this substrate. At low phosphoenolpyruvate levels both isozymes were inhibited by phenylalanine, and half-maximal inhibition was found at 0.3 and 2.2 mm for the kidney and muscle isozymes, respectively. At a 5 mm concentration of the substrate only the kidney isozyme was inhibited, the apparent K_i being the same. Alanine inhibited the kidney isozyme (apparent K_i at 0.3 mm, irrespective of substrate concentration). No effect was seen on the muscle isozyme. Fructose 1,6-bisphosphate was an activator of the kidney isozyme at phosphoenolpyruvate concentrations below 1.0 mm It also counteracted the inhibition by alanine or phenylalanine of this isozyme. ATP inhibited both isozymes, and this inhibition was not counteracted by fructose 1,6-bisphosphate. The kidney isozyme showed both a high and a low apparent K_m for phosphoenolpyruvate in the presence of ATP. The influence of the effectors on the activity of both isozymes varied markedly with pH, except for the action of ATP. At low substrate concentrations, however, the inhibitor action of ATP on the muscle enzyme was diminished around pH 7.5, in contrast to higher or lower pH values. Alanine or phenylalanine were more effective as inhibitors at higher pH values, and fructose 1,6-bisphosphate stimulated the kidney isozyme only at pH levels above pH 6.5. The influence of activators and inhibitors on the regulation of the kidney and muscle pyruvate kinases is discussed.     

Some properties of fructose 1,6-diphosphatase of rat liver and their relation to the control gluconeogenesis

1. Fructose 1,6-diphosphatase has been purified tenfold from rat liver. The final preparation was not contaminated by either glucose 6-phosphatase or phosphofructokinase. The properties of the enzyme have been investigated in an attempt to define factors that could be of revelance to metabolic control of fructose 1,6-diphosphatase activity. 2. The metal ions Fe²⁺, Fe³⁺ and Zn²⁺ inhibited the activity of fructose 1,6-diphosphatase even in the presence of an excess of mercaptoethanol; other metal ions tested had no effect. The inhibition produced by Zn²⁺ was reversed by EDTA, but that produced by either Fe²⁺ or Fe³⁺ was not reversible. 4. The enzyme has a very low K_m for fructose 1,6-diphosphate (2·0μm). Concentrations of fructose 1,6-diphosphate above 75μm inhibited the activity; however, even at very high fructose 1,6-diphosphate concentrations only 70% inhibition was obtained. 5. The activity was also inhibited by low concentrations of AMP, which lowered V_max. and increased K_m for fructose 1,6-diphosphate. Evidence is presented that suggests that AMP can be defined as an allosteric inhibitor of fructose 1,6-diphosphatase. 6. The inhibitions by both fructose 1,6-diphosphate and AMP were extremely specific. Also, the degree of inhibition was not affected by the presence of intermediates of glycolysis, of the tricarboxylic acid cycle, of amino acid metabolism or of fatty acid metabolism. 7. It is suggested that the intracellular concentrations of AMP and fructose 1,6-diphosphate could be of significance in controlling the activity of fructose 1,6-diphosphatase in the liver cell. The possible relationship between these intermediates and the control of gluconeogenesis is discussed.     

Purification and properties of a D-fructose 1,6-bisphosphatase from Saccharomyces cerevisiae.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) from Saccharomyces cerevisiae has been purified to homogeneity. A molecular weight of 115,000 has been obtained by gel filtration. The enzyme appears to be a dimer with identical subunits. The apparent K_m for fructose bisphosphatase varies with the Mg²⁺ concentration of the enzyme, being 1 × 10^?6m at 10 mm Mg²⁺ and 1 × 10^?5m at 2 mm Mg²⁺. Other phosphorylated compounds are not significantly hydrolyzed by the enzyme. An optimum pH of 8.0 is exhibited by the enzyme. This optimum is not changed by addition of EDTA. AMP inhibits the enzyme with a K_i of 8.0 × 10^?5m at 25 °C. The inhibition is temperature dependent, the value of K_i increasing with raising temperature. 2-Deoxy-AMP is also inhibitory with a K_i value at 25 °C of 1.6 × 10^?4m. An ordered uni-bi mechanism has been deduced for the reaction with phosphate leaving the enzyme as the first product and the fructose 6-phosphate as the second one.     

Regulatory sulfhydryl groups, conformational change, and allosteric inhibition in rabbit muscle fructose diphosphatase

The 16 sulfhydryl groups of native, homogeneous rabbit muscle fructose diphosphatase can all react with 5,5′-dithiobis-(2-nitrobenzoic acid). High concentrations of substrate (1–2 mm) decrease the reaction rate of the sulfhydryl groups, while concentrations up to 70 μm have no effect. After titration of the four most rapidly reacting sulfhydryl groups there is a marked desensitization toward the allosteric inhibitor AMP. In the presence of 30 μm AMP only 4–5 sulfhydryl groups/tetramer react with 5,5′-dithiobis-(2-nitrobenzoic acid), and the enzyme again becomes desensitized toward AMP inhibition. Together with a 3.5-fold increase in the I₅₀ for AMP inhibition, the K_m for Mg²⁺ or Mn²⁺ ions is also increased. In the presence of 7 mm MgCl₂ or 0.28 mm MnCl₂ only 6–8 sulfhydryl groups are modified. The rapid reaction of 4 sulfhydryl groups again results in desensitization. There is neither a protection by the substrate against inactivation, nor a protection by the allosteric inhibitor against desensitization. It is concluded that AMP and the divalent cations induce conformational changes in the protein molecule making 11–12 or 8–10 sulfhydryl groups inert for 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. The K_m for fructose-1,6-diphosphate is not changed after the modification of 4–5 sulfhydryl groups.     

Modification of the kinetics and regulatory properties of bovine hepatic fructose 1,6-diphosphatase with pyridoxal 5'-phosphate

Treatment of purified fructose 1,6-diphosphatase from bovine liver (which is maximally active at neutral pH) with pyridoxal 5'-phosphate produces changes in the spectral, catalytic, and allosteric properties of the enzyme. After modification the Michaelis constants for fructose-1,6-diphosphate and Mg²⁺ are increased, and inhibition by AMP is decreased. Substrate inhibition is decreased, but not abolished; the curve is merely shifted toward higher substrate concentration. Fructose-1, 6-diphosphate protects against the increases in the K_m for fructose-1, 6-diphosphate and the K_m for Mg²⁺, and against the changes in substrate inhibition, but not against the changes in AMP inhibition. AMP protects against the changes in AMP inhibition and the increase in the K_m for magnesium, but does not prevent the changes in substrate inhibition or the increase in the K_m for fructose-1, 6-diphosphate. The pH curves in the modified enzyme are altered at high, but not at low, substrate concentration.     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

Isolation and Characterization of Two Enzymes Capable of Hydrolyzing Fructose-1,6-Bisphosphatase from the Lichen Peltigera rufescens

Two enzymes capable of hydrolyzing fructose-1,6-bisphosphate (FBP) have been isolated from the foliose lichen Peltigera rufescens (Weis) Mudd. These enzymes can be separated using Sephadex G-100 and DEAE Sephacel chromatography. One enzyme has a pH optimum of 6.5, and a substrate affinity of 228 micromolar FBP. This enzyme does not require MgCl₂ for activity, and is inhibited by AMP. The second enzyme has a pH optimum of 9.0, with no activity below pH 7.5. This enzyme responds sigmoidally to Mg²⁺, with half-saturation concentration of 2.0 millimolar MgCl₂, and demonstrates hyperbolic kinetics for FBP (K_m = 39 micromolar). This enzyme is activated by 20 millimolar dithiothreitol, is inhibited by AMP, but is not affected by fructose-2-6-bisphosphate. It is hypothesized that the latter enzyme is involved in the photosynthetic process, while the former enzyme is a nonspecific acid phosphatase.     

Crystalline fructose 1,6-bisposphatase from chicken breast muscle.

Fructose 1,6-bisphosphatase has been isolated and crystallized in high yield from chicken breast muscle, which is a rich source of this enzyme. The specific activity assayed at pH 7.4 and 25 °C in the presence of 0.2 mm MnCl₂ 0.1 mm EDTA, and 40 mm ammonium sulfate is 50–60 units/mg, making this one of the most active fructose bisphosphatases yet described. The K_m for fructose bisphosphate is 8.3 μm. AMP (0.4 μm) inhibits the activity at pH 7.4 almost completely. EDTA can be replaced as activator by citrate or histidine, which both give maximum activation at millimolar concentrations. Citrate is as effective as EDTA. The enzyme has a molecular weight of 144,000 and is composed of four subunits having a molecular weight of 36,000. Amino- and carboxy-terminal analyses indicate that the subunits are identical.     

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.     

Purification and characterization of pyruvate kinase from Trypanosoma brucei

Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been partially purified by carboxymethylcellulose chromatography, and gel filtration. The enzyme is unstable in aqueous solution and requires the presence of a thiol protecting reagent as well as glycerol for the maintenance of activity. Dithiothreitol activates as well as stabilizes the enzyme. Phosphoenolpyruvate allosterically activates trypanosome pyruvate kinase whereas hyperbolic kinetics are found when ADP is the variable substrate. Mg²⁺ or Mn²⁺ ions and a monovalent cation are essential for enzyme activity. Fructose 1,6-diphosphate acts as a heterotropic allosteric activator, markedly decreasing the S_0.5 value for phosphoenolpyruvate from 1.34 to 0.25 mm at 1 mm fructose 1,6-diphosphate and transforms the phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form. The enzyme has a pH optimum of 6.5–7.0 and a molecular weight of 270,000 ± 27,000 as estimated by gel chromatography. Purine nucleotides are the preferred coenzymes for the reaction, having much lower K_m values than the pyrimidine nucleotides. The possible role of pyruvate kinase in the regulation of glycolysis in T. brucei is discussed.     

Zn2+, Mg2+, and H+ binding to d-fructose 1,6-bisphosphate studied by 31P and 1H NMR

The anomeric composition and mutarotation rates of fructose 1,6-bisphosphate were determined in the presence of 100 mm KCl at pH 7.0 by ³¹P NMR. At 23 and 37 °C the solution contains (15 ± 1)% of the α anomer. The anomeric rate constants at 37 °C are (4.2 ± 0.4) s^?1 for the β → α anomerization and (14.9 ± 0.5) s^?1 for the reverse reaction. A D₂O effect between 2.1 and 2.6 was found. From acid base titration curves it appeared that the pK values of the phosphate groups range from 5.8 to 6.0. Mg²⁺ and Zn²⁺ bind preferentially to the 1-phosphate in the α-anomeric position. Zn²⁺ has a higher affinity for this phosphate group than Mg²⁺ has. At increasing pH the fraction α anomer decreases slightly. At increasing Mg²⁺/fructose 1,6-bisphosphate ratios the fraction α anomer increases till 19% at a ratio of 20. Proton and probably Mg²⁺ binding decreases the anomerization rate. The time-averaged preferred orientation of the 1-phosphate along the C₁O₁ bond of the α conformer is strongly pH dependent, gauche rotamers being predominant at pH 9.4. In the presence of divalent cations the orientation is biased toward trans. A mechanistic model is proposed to explain the Zn²⁺, Mg²⁺, and pH-dependent behavior of the gluconeogenic enzyme fructose 1,6-bisphosphatase.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Studies on nucleotidases in plants. Reversible denaturation of the crystalline mung bean nucleotide pyrophosphatase and the effect of adenylates on the native and renatured enzyme

The crystalline mung bean nucleotide pyrophosphatase was inhibited nonlinearly by AMP, one of the products of the reaction. The partially inactive enzyme was specifically reactivated by ADP, and V at maximal activation was the same as that of the native enzyme. ATP was a linear, noncompetitive inhibitor. The kinetic evidence suggested that ADP and ATP might not be reacting at the same site as AMP. The electrophoretic mobility of the enzyme was increased by AMP, whereas ADP and ATP were without effect.The enzyme was denatured on treatment with urea or guanidine hydrochloride. The renatured and the native enzyme had the same pH (9.4) and temperature (49 °C) optimum. The K_m (0.2 mm) and V (3.2) of the native enzyme increased on renaturation to 1.8 mm and 8.0, respectively. In addition, renaturation resulted in desensitization of the enzyme to inhibition by low concentrations of AMP. Renaturation did not affect the reactivation of the apoenzyme by Zn²⁺.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Activation of ribulose 1,5-diphosphate carboxylase by nicotinamide adenine dinucleotide phosphate and other chloroplast metabolites

Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 10 mm MgCl₂ and 1 mm bicarbonate in the absence of ribulose 1,5-diphosphate, can be further activated about 170% with 0.5 mm NADPH present in the preincubation mixture. NADP⁺, NADH, and NAD⁺ are ineffective. The activation by NADPH is comparable to that previously seen with 0.05 to 0.10 mm 6-phosphogluconate in that these specific preincubation conditions are required, but the effects of NADPH and 6-phosphogluconate are not additive. Moreover, where higher concentrations of 6-phosphogluconate inhibited the enzyme, higher concentrations of NADPH give a greater activation, saturating at about 1 mm and 200%. Under the specified conditions of preincubation, fructose 1,6-diphosphate has an activation curve similar to that of 6-phosphogluconate, peaking at 0.1 mm and 70%. Above this level, activation decreases, and inhibition is seen at still higher concentrations. Other metabolites tested produced smaller or no effects on the enzyme activity assayed under these conditions. When either reduced NADP or 6-phosphogluconate are present in the preincubation mixture, it becomes possible to determine the Km for bicarbonate using a Lineweaver-Burk plot, and the Km for bicarbonate under these conditions is 2.8 mm, corresponding to 0.3% CO₂ at pH 7.8 and 25 C.     

Further studies on the properties of the rabbit reticulocyte adenosine 3',5'-cyclic monophosphate-dependent protein kinase I

The properties of cyclic AMP-dependent protein kinase I isolated from rabbit reticulocytes were further investigated. The enzyme catalyzes the phosphorylation of histone in the presence of ATP and Mg²⁺ and this reaction is stimulated by cyclic AMP. The pH optimum of the reaction was between 8.5 and 9.0, when assayed in the presence of cyclic AMP. No distinct pH optimum was observed in the absence of the cyclic nucleotide. The K_m values for ATP appeared to be very similar whether it was determined in the presence (K_m = 1.7 × 10⁻⁴m) or absence (K_m = 2.5 × 10⁻⁴m) of cyclic AMP. The rate of heat inactivation of the catalytic activity and the cyclic AMP binding activity of kinase I were found to be dependent on the presence of Mg²⁺, ATP, and/or cyclic AMP. In the presence of cyclic AMP, the rate of inactivation of the catalytic activity of kinase I at 53 ° was accelerated. On the other hand, the cyclic AMP binding activity appeared to be protected from heat inactivation by the cyclic nucleotide. When both ATP and Mg²⁺ were present in the heating mixture, no loss of catalytic and binding activities of kinase I were observed even up to 8 min of heating at 53 °. The cyclic AMP binding activity of kinase I was almost completely inhibited by mercuric acetate at a concentration of 1 mm, while the loss in catalytic activity was only 50%. These results substantiate our previous observation that kinase I contains two nonidentical subunits, a catalytic subunit and a cyclic AMP binding subunit.     

Oxygenase properties of crystallized fraction 1 protein from tobacco.

Ribulose 1,5-diphosphate-dependent oxygenase activity was demonstrated for crystallized Fraction 1 protein (RuDP² carboxylase EC 4.1.1.39) from tobacco. The kinetic properties of this oxygenase function were examined polarographically in air-equilibrated medium. Optimum activity was obtained at pH 8.4–8.6, and required 4–8 mm MgCl₂. Higher Mg²⁺ concentrations decreased activity and slightly shifted the pH optimum to 8.2–8.3. The apparent K_m (RuDP) and K_m (Mg²⁺) were 22 μm and 0.5 mm, respectively. Oxygenase activity was inhibited by bicarbonate and indirectly by KCN. Kinetic studies suggest that the active inhibitory substance is the cyanohydrin derivative formed from the reaction of KCN with RuDP.Changes in oxygenase kinetics were observed upon addition of RuDP, as previously reported for the carboxylase function of this enzyme. Oxygenase activity required preincubation of the enzyme with both Mg²⁺ and low concentrations of bicarbonate. Activities were enhanced about 20 and 70% when FDP (0.1 mm) and NADPH (0.5 mm), respectively, were included during preincubation.     

Xylulose 1,5-Bisphosphate Synthesized by Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase during Catalysis Binds to Decarbamylated Enzyme

Xylulose 1,5-bisphosphate (XuBP) is synthesized from ribulose 1,5-bisphosphate (RuBP) at carbamylated catalytic sites on ribulose 1,5-bisphosphate carboxylase (Rubisco) with significant amounts of XuBP being formed at pH less than 8.0. XuBP has been separated by high performance liquid chromatography and identified by pulsed amperometry from compounds bound to Rubisco during catalysis with the purified enzyme and from celery (Apium graveolens var Utah) leaf extracts. XuBP does not bind tightly to carbamylated sites, but does bind tightly to decarbamylated sites. Upon incubation of fully activated Rubisco with 5 micromolar XuBP, loss of activator CO₂ occurred before XuBP bound to the enzyme catalytic sites, even in the presence of excess CO₂ and Mg²⁺. Binding of XuBP to decarbamylated Rubisco sites was highly pH dependent. At pH 7.0 and 7.5 with 10 millimolar MgCl₂ and 10 millimolar KHCO₃, the apparent dissociation constant for XuBP, K_d, was 0.03 micromolar, whereas at pH 8.0 and 8.5, the apparent K_d was 0.35 and 2.0 micromolar, respectively. This increase in K_d with pH was a result of a decrease in the association rate constant and an increase in the dissociation rate constant of XuBP bound to decarbamylated sites on Rubisco. The K_d of 2-carboxyarabinitol 1-phosphate binding to carbamylated sites was only slightly pH dependent.     

Hysteretic behavior of rat liver fructose 1,6-bisphosphatase induced by zinc ions

The measurement of the time dependency of the activity of rat liver fructose 1,6-bisphosphatase shows that the enzyme under certain conditions exhibits kinetic hysteretics. After addition of the substrate, the enzyme is initially in a state characterized by a “high” K_m of about 2 μm. During the reaction the enzyme is converted in a slow process to a low K_m form (K_m is about 0.5 μm). The transition is accompanied by a decrease in V. It is concluded that the hysteretic behavior is caused by binding of the Zn²⁺ substrate complex to the enzyme. The earlier reported effect of glucagon treatment on the activity of fructose 1,6-bisphosphate (O. D. Taunton, F. B. Stifel, H. L. Greene, and R. H. Herman (1974) J. Biol. Chem.249, 7228–7239) was reinvestigated, taking into account the hysteretic behavior. Under conditions where the pyruvate kinase activity is decreased by glucagon injection, no activity change of fructose 1,6-bisphosphatase is observed. It can be suggested that for studies concerning the effects of incubation or hormone treatment on fructose 1,6-bisphosphatase, the complex kinetics of the rat liver enzyme has to be taken into account.