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.     

Characterization of phosphoenolpyruvate carboxylase from mature maize seeds: Properties of phosphorylated and dephosphorylated forms

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from mature maize seeds (Zea mays L.) was purified to homogeneity and a final specific activity of 13.3 μmol min⁻¹ mg⁻¹. Purified PEPC was treated with phosphatase from bovine intestinal mucosa or protein kinase A to study its apparent phosphorylation level. Kinetic parameters of the enzyme reaction catalyzed by phosphorylated and dephosphorylated forms under different conditions were compared, as well as an effect of modulators. The enzyme dephosphorylation resulted in the change of hyperbolic kinetics to the sigmoidal one (with respect to PEP), following with the decrease of maximal reaction rate and the increase of sensitivity to l-malate inhibition. The hyperbolic kinetics of native PEPC present in dry maize seeds was not changed after the protein kinase A treatment, while it was converted to the sigmoidal one after dephosphorylation. Level of PEPC phosphorylation was not affected during seed imbibition.     

Effects of high concentration of salts on the esterase activity and structure of a kiwifruit peptidase, actinidain

Effects of salts on the activity and stability of actinidain were examined. With increasing salt concentration up to 0.5 M, the activity (kcat/Km) for N-alpha-Cbz-L-lysine p-nitrophenyl ester decreased to 40% of that in the absence of salt. The inhibitor constant Ki of LiCl, NaCl, and KCl was 0.16-0.43 M. With 3 M KCl and NaCl, the specificity constant kcat/Km recovered to 110 and 75%, respectively. No re-activation was observed with LiCl. The inhibition and re-activation were dependent on the changes in both Km and kcat, whereas no CD change was observed. The tryptophan fluorescence of actinidain was not affected by 0-0.5 M salt, but a considerable decrease in its intensity was observed with increasing salt concentration from 0.5 to 3.0 M. These results suggest that the inhibition observed with the lower salt concentration (<0.5 M) is due to attenuation of the electrostatic interaction between the enzyme and substrate, and the higher concentration (0.5-3.0 M) induces structural change in the states of tryptophan residues, which is associated with the re-activation. Actinidain keeps considerably high activity and stability even in the presence of 3 M salts.     

Characterization of NADP-Isocitrate Dehydrogenase from Nodules of Pigeonpea (Cajanus cajan)

NADP-isocitrate dehydrogenase from nodules of pigeonpea (Cajanus cajan L. cv UPAS-120) was partially purified to about 57 folds and its properties were studied. The enzyme showed an absolute requirement for a divalent cation which was fulfilled either by Mn⁺² or Mg⁺² and to a smaller extent by Co⁺². The enzyme exhibited a sigmoidal response to increasing concentrations of Mn²⁺ (S_0.5=0.3mM). The apparent Km values for isocitrate, NADP and Mg²⁺ were 21, 23 and 280 μM, respectively. It had an optimum pH of 8.0–8.2. The enzyme activity was not affected by various organic acids, amino acids and amides. NADH inhibited the activity non-competitively with respect to NADP. An apparent inhibition by ATP and ADP was due to chelation of divalent cation. NADPH acted competitively against NADP and non-competitively against isocitrate. Glutamate caused uncompetitive inhibition with respect to NADP and competitive against isocitrate. Kinetic studies suggested the reaction mechanism to be probably random sequential. Possible regulation of the enzyme activity in the nodules via cellular redox state and the levels of reaction products is discussed.     

Heparin solubilizes asymmetric acetylcholinesterase from rat neuromuscular junction

Citrate synthase from Acinetobacter calcoaceticus was subjected to proteolysis with subtilisin. Although the enzyme proved relatively resistant to inactivation by this treatment, SDS-polyacrylamide gel electrophoresis clearly revealed breakdown of the citrate synthase to smaller fragments. The regulatory responses of the native enzyme to inhibition by NADH and re-activation by AMP were retained on proteolysis, indicating that the fragments bind tightly to each other and preserve the overall cooperative molecular interactions.     

A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum.

1. On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction. The rate became approximately constant after declining to 4-5% of the initial value. Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate. The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH. 2. Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution. At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min. 3. Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy. 4. 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective. 5. The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation. 6. Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8. 7. The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates. 8. The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues.     

Studies on nucleotidases in plants. Dimerization of the crystalline mung bean nucleotide pyrophosphatase by 5'-adenosine monophosphate and the properties of the dimerized enzyme.

The addition of AMP to the crystalline and homogeneous mung bean nucleotide pyrophosphatase [EC 3.6.1.9]altered its electrophoretic mobility. AMP was tightly bound to the enzyme and was not removed on passage through a column of Sephadex G-25 or on electrophoresis. The molecular weight of the native and AMP-modified enzymes were 65,000 and 136,000, respectively. The properties of the native enzyme such as the pH (9.4) and temperature (49 °C) optima, inhibition by EDTA, reversal of EDTA-inhibition by Zn²⁺ and Co²⁺, were not altered on dimerization by AMP. The AMP-modified enzyme had a linear time-course of reaction, unlike the native enzyme which exhibited a biphasic time-course of reaction. The AMP-modified enzyme was irreversibly denatured by urea. AMP concentrations larger than 100 μM inhibited linearly the activity of the AMP-modified enzyme. ADP and ATP inhibited the activity in a sigmoidal manner. K_m and V of the native and AMP-modified enzymes were, 0.25 mm and 0.58 mm; and 3.3 and 2.5, respectively.     

Activity of reduced ubiquinone: Cytochrome c oxidoreductase with various ubiquinol-isoprenologues as substrate and corresponding inhibitory effect of antimycin in yeast

1. Reduced ubiquinones-1, -2, -3, -4 and -6 were used as substrates for ubiquinol: cytochrome c oxidoreductase.2. The portion of antimycin-sensitive activity depends on the concentration of ubiquinol and on the pH. Only reduced ubiquinone-2 and reduced ubiquinone-3 show high activities the main part of which is sensitive to antimycin.3. The antimycin effect curve of ubiquinol: cytochrome c oxidoreductase is linear in shape with reduced ubiquinone-2 as substrate but sigmoidal with reduced ubiquinone-3 and succinate. Ubiquinol-3: cytochrome c oxidoreductase activity contains a portion scarcely affected by antimycin. About 300 pmoles of antimycin per mg protein, enough to inhibit succinate, NADH- and reduced ubiquinone-2:cytochrome c oxidoreductase almost totally, affect ubiquinol-3: cytochrome c oxidoreductase to only about 80% and another 300 pmoles of antimycin are needed for the next 10% of inhibition.4. The activities of succinate- and NADH: cytochrome c oxidoreductase are stimulated by ubiquinones-2 and -3. The shapes of the inhibition curves by antimycin of the stimulated activities are sigmoidal. About twice the amount of antimycin is necessary to inhibit stimulated activities to the same value as the unstimulated.5. The non-ionic detergent Lubrol WX is not effective in stimulating enzymatic activities. However, in the presence of 0.6 M sorbitol, it converts the linear antimycin effect curve with reduced ubiquinone-2 as substrate, into sigmoidal.6. NADH- and succinate: cytochrome c oxidoreductase activities and reduced ubiquinone-2 and reduced ubiquinone-3: cytochrome c oxidoreductase activities become deactivated with increasing concentrations of the non-ionic detergent Lubrol WX. The activity with reduced ubiquinone-2 as substrate is less resistant to the action of the detergent than with reduced ubiquinone-3. The b-cytochromes do not become CO-reactive by this treatment.7. Deoxycholate in low concentrations does not stimulate ubiquinol: cytochrome c oxidoreductase activity. It converts the inhibition curve by antimycin from sigmoidal to linear with increasing concentrations of the detergent with all substrates tested. The amount of antimycin needed for 90% inhibition of reduced ubiquinone activities is about the same under these conditions as with succinate, NADH or reduced ubiquinol in untreated particles.8. The results are discussed with respect to the theories of the electron transport mechanism and of the inhibition by antimycin of the electron flow through the bc₁-segment of the respiratory chain in beef heart.     

MgATP-induced inhibition of the adenosine triphosphatase activity of the chloroform-released mitochondrial adenosine triphosphatase.

1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined.     

Kinetics of cytochrome c oxidase from yeast. Membrane-facilitated electrostatic binding of cytochrome c showing a specific interaction with cytochrome c oxidase and inhibition by ATP (With an appendix on the occurrence of two-dimensional diffusion of cytochrome c on the membrane surface)

The steady-state kinetics of purified yeast cytochrome c oxidase were investigated at low ionic strength where the electrostatic interaction with cytochrome c is maximized. In 10 mM cacodylate/Tris (pH 6.5) the oxidation kinetics of yeast iso-1-cytochrome c were sigmoidal with a Hill coefficient of 2.35, suggesting cooperative binding. The half-saturation point was 1.14 μM. Horse cytochrome c exhibited Michaelis-Menten kinetics with a higher affinity (K_m = 0.35 μM) and a 100% higher maximal velocity.In 67 mM phosphate the Hill coefficient for yeast cytochrome c decreased to 1.42, and the species differences in Hill coefficients were lessened. Under the latter conditions, a yeast enzyme preparation partially depleted of phospholipids was activated on addition of diphosphatidylglycerol liposomes. When the enzyme was incorporated into sonicated yeast promitochondrial particles the apparent K_m for horse cytochrome c was considerably lower than the value for the isolated enzyme.ATP was found to inhibit both the isolated oxidase and the membrane-bound enzyme. With the isolated enzyme in 10 mM cacodylate/Tris, 3 mM ATP increased the half-saturation point with yeast cytochrome c 3-fold, without altering the maximal velocity or the Hill coefficient. 67 mM phosphate abolished the inhibition of the isolated oxidase by ATP.The increase in affinity for cytochrome c produced by binding the oxidase to the membrane was not observed in the presence of 3 mM ATP, with the result that the membrane-bound enzyme was more sensitive to inhibition by ATP. ADP was a less effective inhibitor than ATP, and did not prevent the inhibition by ATP.It is proposed that non-specific electrostatic binding of cytochrome c to phospholipid membranes, followed by rapid lateral diffusion, is responsible for the dependence of the affinity on the amount and nature of the phospholipids and on the ionic strength.ATP may interfere with the membrane-facilitated binding of cytochrome c by a specific electrostatic interaction with the membrane or by binding to cytochrome c.     

Structural Basis for Certain Naturally Occurring Bioflavonoids to Function as Reducing Co-Substrates of Cyclooxygenase I and II

Background

Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds.

Methodology/Principal Findings

A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG₂ to PGE₂, a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.

Conclusions/Significance

These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation.     

Kinetic characterization of spinach leaf sucrose-phosphate synthase

The spinach (Spinacia oleracea) leaf sucrose-phosphate synthase was partially purified via DEAE-cellulose chromatography, and its kinetic properties were studied. Fructose-6-phosphate saturation curves were sigmoidal, while UDPglucose saturation curves were hyperbolic. At subsaturating concentrations of fructose-6-phosphate, 1,5 anhydroglucitol-6-phosphate had a stimulatory effect on enzyme activity, suggesting multiple and interacting fructose-6-phosphate sites on sucrose-phosphate synthase. The concentrations required for 50% of maximal activity were 3.0 millimolar and 1.3 millimolar, respectively, for fructose-6-phosphate and UDPglucose. The enzyme was not stimulated by divalent cations. Inorganic phosphate proved to be a potent inhibitor, particularly at low concentrations of substrate. Phosphate inhibition was competitive with UDPglucose, and its K_i was determined to be 1.75 millimolar. Sucrose phosphate, the product of the reaction, was also shown to be a competitive inhibitor towards UDPglucose concentration and had K_i of 0.4 millimolar. The kinetic results suggest that spinach leaf sucrose-phospahte synthase is a regulatory enzyme and that its activity is modulated by the concentrations of phosphate, fructose-6-phosphate, and UDPglucose occurring in the cytoplasm of the leaf cell.     

The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K_0.5 for fructose-6-P and a decrease in the apparent k_cat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n_H of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.     

Biphasic kinetics of metal ion reactivation of trypsin-thiol complexes

This report describes biphasic kinetic data obtained when trypsin was inhibited by a thiol-containing inhibitor present in Ehrlich ascites tumour cells and then subjected to addition of Hg2+, Cu2+ or Ag+. This resulted in an initial re-activation of the trypsin, followed by inhibition of the enzyme with the addition of higher concentrations of these ions. The significance of these observations is 2-fold: (i) help to elucidate the mechanism of metal ion activation of latent enzymes, and (ii) also indicate that, in certain circumstances, the concentration of added metal ion determines whether the metal acts as an activator or an inhibitor of enzyme activity.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl posphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at near-neutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membranes. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.     

New insights into the catalytic mechanism of human glycine N‐acyltransferase

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC–ESI‐MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi–Bi and/or ping‐pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl‐CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis–Menten reaction mechanism.     

Properties of phosphofructokinase 1 fromPythium ultimum andAgaricus bisporus and comparison with other fungal phosphofructokinases 1

Phosphofructokinase 1 was partially purified fromAgaricus bisporus fruit bodies andPythium ultimum mycelium. The enzyme fromA. bisporus was similar to other fungal phosphofructokinases 1: it was inhibited by ATP, had sigmoidal kinetics toward fructose-6-phosphate, and was stimulated by fructose-2,6-bisphosphate. Phosphoenolpyruvate was inhibitory, and phosphate, but not AMP, stimulated activity. The enzyme fromA. bisporus was very unstable. Phosphofructokinase 1 from the OomycetePythium ultimum on the contrary had hyperbolic kinetics to both its substrates and was not activated by fructose-2,6-bisphosphate. The only effectors found were phosphoenolpyruvate, which inhibited, and phosphate, which slightly stimulated enzyme activity. These properties were compared with those of phosphofructokinases 1 from other fungi.     

Kinetic manifestations of allosteric interactions in models of regulatory enzymes with "indirect" co-operativity

The shape of the plots of initial reaction rate (ν) versus initial substrate concentration ([S]₀) and versus initial concentration of allosteric effector ([F]₀) for the model of allosteric enzyme of Monod, Wyman &; Changeux (1965) and for the model of dissociating regulatory enzyme has been analysed by means of the inconstant exponent (q) for substrate or effector concentration, respectively. It has been shown that allosteric interactions in above-mentioned models with “indirect” co-operativity may be manifested not only by the sigmoidal shape of the plot of ν versus [S]₀ or ν versus [F]₀ (with one point of inflexion) but also by the increase in the magnitude of exponent q in progress of saturation process of the enzyme by the substrate or by the effector in the absence of the sigmoidal shape of these plots. It has been shown also that the plot of ν versus [S]₀ has two inflexion points when the parameters have certain definite values. One of these inflexion points (or even both at definite values of the parameters) is hardly discernible. At certain definite values of the parameters two inflexion points may be kinetically manifested by such phenomenon as “negative” co-operativity (q < 1). This is possible if one of the interconvertable enzyme forms exceeds another not only in the affinity to the substrate but also in the value of the rate constant for catalytic breakdown of the enzyme-substrate complex.     

Effect of a highly dispersed zinc powder on the cAMP phosphodiesterase activity in mouse thymocytes

The inhibition of cAMP phosphodiesterase activity by high dispersed zinc powder and zinc ions has been found in vitro experiments. The enzyme activity dependence on concentration of inhibitors is characterized by sigmoidal curve with Hill coefficients 1,8-2,1 and 1,2-1,3 correspondingly that may indicate the presence of positive cooperativity on inhibitor in enzyme. Magnesium ions influence the inhibited effect of zinc ions and the preparation of high dispersed zinc powder.     

Regulation of ADP-Glucose Pyrophosphorylase from Chlorella vulgaris

ADP-glucose pyrophosphorylase was partially purified from Chlorella vulgaris 11h. 3-Phosphoglycerate activated the enzyme by lowering the Michaelis constant for glucose-1-phosphate (from 0.97 to 0.36 millimolar in the presence of 2 millimolar phosphoglycerate) and ATP (from 0.23 to 0.10 millimolar), as well as increasing the V_max. Saturation curves for 3-phosphoglycerate were hyperbolic and the activator concentration at half V_max value for 3-phosphoglycerate was 0.41 millimolar either in the presence or absence of phosphate. Phosphate inhibited the enzyme in a competitive manner with respect to glucose-1-phosphate, but did not affect the Michaelis constant value for ATP. 3-Phosphoglycerate changed neither the inhibitor concentration at half V_max value of 1.0 millimolar for phosphate nor the hyperbolic inhibition kinetics for phosphate. The enzyme required divalent cations for its activity. The activation curves for Mn²⁺ and Mg²⁺ were highly sigmoidal. The activator concentration at half V_max values for Mn²⁺ and Mg²⁺ were 2.8 and 3.7 millimolar, respectively. With optimal cations, the Michaelis constant values for ATP-Mn and ATP-Mg were 0.1 and 0.4 millimolar, respectively.