The Effect of Adenine Nucleotides on Purified Phosphoenolpyruvate Carboxylase from the CAM Plant Crassula argentea

The effects of adenine nucleotides on phosphoenolypyruvate carboxylase were investigated using purified enzyme from the CAM plant, Crassula argentea. At 1 millimolar total concentration and with limiting phosphoenolpyruvate, AMP had a stimulatory effect, lowering the K_m for phosphoenolpyruvate, ADP caused less stimulation, and ATP decreased the activity by increasing the K_m for phosphoenolpyruvate. Activation by AMP was not additive to the stimulation by glucose 6-phosphate. Furthermore, AMP increased the K_a for glucose 6-phosphate. Inhibition by ATP was competitive with phosphoenolpyruvate. In support of the kinetic data, fluorescence binding studies indicated that ATP had a stronger effect than AMP on phosphoenolpyruvate binding, while AMP was more efficient in reducing glucose 6-phosphate binding. As free Mg²⁺ was held constant and saturating, these effects cannot be ascribed to Mg²⁺ chelation. Accordingly, the enzyme response to the adenylate energy charge was basically of the “R” type (involving enzymes of ATP regenerating sequences) according to D. E. Atkinson's (1968 Biochemistry 7: 4030-4034) concept of energy charge regulation. The effect of energy charge was abolished by 1 millimolar glucose 6-phosphate. Levels of glucose 6-phosphate and of other putative regulatory compounds of phosphoenolpyruvate carboxylase were determined in total leaf extracts during a day-night cycle. The level of glucose 6-phosphate rose at night and dropped sharply during the day. Such a decrease in glucose 6-phosphate concentration could permit an increased control of phosphoenolpyruvate carboxylase by energy charge during the day.     

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.     

Two forms of phosphoinositol kinase from germinating mung bean seeds

Phosphoinositol kinase, the key enzyme responsible for the biosynthesis of higher inositol phosphates has been isolated from the cotyledons of mung beans germinated for 24 hr and has been resolved into two different forms, phosphoinositol kinase A and phosphoinositol kinase B. Both forms were purified to homogeneity and characterized. The K_m values for ATP with phosphoinositol kinase A (1.78 × 10^?4 M) and phosphoinositol kinase B (3.12 × 10 ^?5 M) showed that phosphoinositol kinase B had a greater affinity for ATP. ATP could be partially replaced as phosphate donor by UTP and phosphoenolpyruvate in the case of phosphoinositol kinase A but not in the case of phosphoinositol kinase B.     

Purification and properties of pyruvate kinase from Mycobacterium smegmatis

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S_0.5v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest K_m value. The enzyme showed an absolute requirement for divalent cations (either Mg²⁺ or Mn²⁺), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg²⁺-activated enzyme to varying degrees (Ni²⁺ > Zn²⁺ > Cu²⁺ > Ca²⁺ > Ba²⁺). The differences in the kinetic responses of the enzyme to Mg²⁺ and Mn²⁺ are discussed.     

Canine erythrocyte pyruvate kinase. II. Properties of the abnormal enzyme associated with hemolytic anemia in the basenji dog

We have compared the solubility, kinetic, immunological, and electrophoretic properties of erythrocyte pyruvate kinase from normal dogs and Basenji dogs with congenital hemolytic anemia due to pyruvate kinase deficiency. Differences can be detected between the two enzymes by all methods. The enzyme from the affected animals has a greater solubility in ammonium sulfate. It has a lower K _m for phosphoenolpyruvate, while the K _m for ADP is increased. This enzyme is not inhibited by ATP or activated by fructose 1,6-diphosphate. The enzyme from the affected animals has none of the allosteric properties characteristic of the normal canine enzyme. No difference can be detected by enzyme inactivation with rabbit antiserum against the human erythrocyte enzyme, but a slight spur is observed on comparison of the two enzymes by Ouchterlony immunodiffusion. The enzymes also differ in their electrophoretic mobilities on starch gel electrophoresis.     

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 kinetic properties of pyruvate kinase from Phycomyces blakesleeanus

• 1.1. Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555(−) has been partially purified and some kinetic properties has been investigated at pH 7.5.
• 2.2. Positive homotropic interactions were observed with phosphoenolpyruvate and Mg²⁺, showing Hill coefficient values of 2.8 and 2.5, respectively, whereas hyperbolic kinetics are found when ADP was the variable substrate.
• 3.3. Fructose 1,6-bisphosphate acts as a heterotropic allosteric activator, markedly decreasing the S_0.5 value for phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form.
• 4.4. ATP inhibits pyruvate kinase from mycelium of Phycomyces blakesleeanus. ATP appears to be a non-competitive inhibitor with respect PEP and competitive inhibitor with respect ADP.

     

Gas chromatographic measurement of urinary 5-fluoro-2'-deoxyuridine levels after barium salt precipitation and sephadex LH-20 cleanup

A fluorescent photoaffinity label—8-azido-1-N⁶-etheno-adenosine 5′-triphosphate (8-N₃ε ATP)—for ATP-binding proteins has been synthesized. The effectiveness of the label is demonstrated with F₁ATPase from Micrococcus luteus. 8-N₃ε ATP is a substrate for the enzyme in the presence of bivalent cations. Ultraviolet irradiation of F₁ATPase in the presence of the label and Mg²⁺ ions inhibits the enzyme irreversibly. The fluorescent label is bound preferentially to the β subunit of the enzyme. Labeling and inactivation are decreased by protection with ATP or ADP.     

A microsomal ATP-activated pyridine nucleotide transhydrogenase

An ATP-activated transhydrogenase which catalyzes the reduction of TPN⁺ by DPNH has been demonstrated in the microsomal fraction from the endosperm of immature Echinocystis macrocarpa seeds. The activity is specifically dependent on the presence of ATP (K_m of approximately 0.1 mm) of several nucleotides tested. The reaction is stimulated by MgCl₂ addition up to concentrations of 6 mm. When 10^?2m EDTA is added to the assay mixture in the absence of added MgCl₂, a transhydrogenation reaction is observed which no longer shows any dependence on added ATP. A TPN⁺-dependent ATPase activity can be demonstrated in these preparations, but no fixed stoichiometry between ATP cleavage and TPNH formation could be established. A lag in attaining the maximal rate of transhydrogenation is seen unless the enzyme is preincubated for 10 min with ATP before initiating the reaction. It can further be shown that preincubation of the enzyme with ATP followed by removal of the ATP on a Dowex 1 column produces an enzyme capable of catalyzing the transhydrogenation without the further addition of ATP. 2,4-Dinitrophenol and thyroxin are effective inhibitors of the transhydrogenase and 2,4-dinitrophenol was shown to inhibit the activating effect of ATP during the preincubation period. It is concluded that the role of ATP is in the modification of the enzyme rather than direct participation in the transhydrogenation. The transhydrogenase is inhibited by ADP and AMP. This results in a response of the enzyme to adenylate energy charge in a manner characteristic for regulatory enzymes which participate in ATP-utilizing metabolic sequences.     

Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm

Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK_c, is localized in the cytosol, and the other, PK_p, is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K⁺ and Mg²⁺. Both isozymes are inhibited by citrate, pyruvate, and ATP. PK_c has a much broader pH profile than PK_p and is also more stable. Both have the same K_m (0.05 millimolar) for PEP, but PK_p has a 10-fold higher K_m (0.3 millimolar) for ADP than PK_c (0.03 millimolar). PK_c also has a higher affinity for alternate nucleotide substrates than PK_p. The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue.     

Sulphate activation in the unicellular red alga Rhodella

ATP-sulphurylase (ATP: sulphate adenylyl-transferase, E.C. 2.7.74) from the unicellular red alga Rhodella has been purified 14-fold by (NH₄)₂SO₄ fractionation. It exhibits a temperature optimum of 31°, an activation energy of 10.8 kcal, has a pH optimum between 7.5 and 9.0 and forms unstable intermediates when incubated with ATP and group VI anions (CrO₄^2?, MoO₄^2?), WO₄^2?), resulting in the accumulation of pyrophosphate. Of the nucleotides tested, only ATP is acted upon by the enzyme. A divalent ion is required for activity and stimulation of the enzyme is 5 times higher with Mg²⁺ than any other ion tested. The actual substrate for the reaction is a Mg-ATP^2? complex. Free ATP inhibits the reaction. APS-[³⁵S] and traces of PAPS-[³⁵S] are formed when cell-free extract from Rhodella is incubated with ATP and sulphate-[³⁵S]. This indicates the existence of APS-kinase (ATP:adenylyl-sulphate 3¹-phosphotransferase, E.C. 2.7.1.25) as well as ATP-sulphurylase.     

Spinach leaf phosphoenolpyruvate carboxylase: purification, properties, and kinetic studies

Spinach leaf phosphoenolpyruvate carboxylase has been purified to homogeneity using salt fractionatjon, chromatography, and immunologie procedures to remove contaminating ribulose diphosphate carboxylase. From gel filtration and isoelectric focusing, the molecular weight (~560,000) and isoelectric point (pI = 4.9) are indistinguishable from those of ribulose diphosphate carboxylase. The subunit molecular weight of phosphoenolpyruvate carboxylase (130,000) suggests that the native enzyme is a tetramer.Kinetic studies using Mg²⁺ or Mn²⁺ as the activator indicate that the divalent cation lowers the K_m of the substrate phosphoenolpyruvate by an order of magnitude and conversely, that the presence of the substrate similarly lowers the K_m of the metal ion, suggesting an enzyme-metal-substrate bridge complex. Three analogs of phosphoenolpyruvate, lphospholactate, d-phospholactate, and phosphoglycolate are potent competitive inhibitors. The inhibitor constant (K_i) of l-phospholactate (2 μm) is 49-fold lower with Mn²⁺ as the activator than with Mg²⁺. An analysis of the competitive inhibition by portions of the l-phospholactate molecule (i.e., l-lactate, methyl phosphate, and phosphite) indicates this 49-fold lowering is due to increased interaction of the phosphoryl group and, to a lesser extent, of the carboxyl and C-O-P bridge oxygen of l-phospholactate with the enzyme metal complex. The results provide indirect evidence for phosphoryl coordination by the enzyme-bound divalent cation.     

Purification and properties of enolase from swine kidney

The first, enolase (2-phospho-d-glycerate hydrolyase, EC 4.2.1.11) to be isolated from a gluconeogenic tissue, swine kidney, was purified more than 600-fold to near homogeneity, as estimated from sedimentation equilibrium and velocity measurements and from disc electrophoresis patterns. The physical properties of the enzyme were examined. Purified kidney enolase has a s^0.87%_20,w = 5.87 S, M_r = 90,000 ± 4,500, e^0.1%_280,1cm = 1.07/mg/ml, a Stokes radius of 37.0 Å, and an apparent subunit molecular weight of 52,000.The amino acid composition was determined and compared with those of mammalian muscle enolases. The partial specific volume calculated from the amino acid composition was found to be 0.728 cc/g. Swine kidney enolase had 12 cysteines per mole; in the native enzyme, two reacted with DTNB.The enzyme was stabilized by magnesium, sucrose, or glycerol; activity lost, on prolonged storage could be completely recovered by treatment with mercaptoethanol and EDTA at 37 °C. Some evidence was obtained for the existence of active monomers of this enzyme. This form of swine kidney enolase was quite unstable, however. The pH optimum was at 6.8. The Michaelis constants for 2-phospho-d-glycerate and phosphoenolpyruvate were 5.10^?5m and 10^?4m; that for magnesium was 4.10^?4m. Substrate inhibition was found for 2-phosphoglycerate but not for phosphoenolpyruvate. No inhibition is seen under comparable conditions with mammalian enolases from glycolytic tissues. This finding is discussed.     

Photosynthetic activities of isolated bundle sheath cells in relation to differing mechanisms of C-4 pathway photosynthesis.

Photosynthetic activities of bundle sheath cell strands isolated from several C₄ pathway species were examined. These included species that decarboxylate C₄ acids via either NADP-malic enzyme (Zea mays, NADP-malic enzyme-type), NAD-malic enzyme (Atriplex spongiosa and Panicum miliaceum, NAD-malic enzyme-type) or phosphoenolpyruvate carboxykinase (Chloris gayana and Panicum maximum, phosphoenolpyruvate carboxykinase-type). Preparations from each of these species fixed ¹⁴CO₂ at rates ranging between 1.2 and 3.5 μmol min^?1 mg^?1 of chlorophyll, with more than 90% of the ¹⁴C being assimilated into Calvin cycle intermediates. With added HCO₃^? the rate of light-dependent O₂ evolution ranged between 2 and 4 μmol min^?1 mg^?1 of chlorophyll for cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species but with Z. mays cells there was no O₂ evolution detectable. Most of the ¹⁴CO₂ fixed by Z. mays cells provided with H¹⁴CO₃^? plus ribose 5-phosphate accumulated in the C-1 of 3-phosphoglycerate. However, 3-phosphoglycerate reduction was increased several fold when malate was also provided. Cells from all species rapidly decarboxylated C₄ acids under appropriate conditions, and the CO₂ released from the C-4 carboxyl was reassimilated via the Calvin cycle. Malate decarboxylation by Z. mays cells was dependent upon light and an endogenous or exogenous source of 3-phosphoglycerate. Bundle sheath cells of NAD-malic enzyme-type species rapidly decarboxylated [¹⁴C]malate when aspartate and 2-oxoglutarate were also provided, and [¹⁴C]aspartate was decarboxylated at similar rates when 2-oxoglutarate was added. Cells from phosphoenolpyruvate carboxykinase-type species decarboxylated [¹⁴C]aspartate when 2-oxoglutarate was added and they also catalyzed a slower decarboxylation of malate. Cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species evolved O₂ in the light when C₄ acids were added. These results are discussed in relation to proposed mechanisms for photosynthetic metabolism in the bundle sheath cells of species utilizing C₄ pathway photosynthesis.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.     

Purification and characterization of myo-inositol hexaphosphate-adenosine diphosphate phosphotransferase from Phaseolus aureus

myo-Inositol hexaphosphate adenosine diphosphate phosphotransferase transfers phosphate from myo-inositol hexaphosphate to adenosine diphosphate to synthesize adenosine triphosphate. This enzyme has been isolated and purified from ungerminated mungbean seeds and found to be different from guanosine diphosphate phosphotransferase. A purification of about 200-fold with 15% recovery has been obtained. The optimal pH of the reaction is 7.0 and is dependent on the presence of a divalent cation, i.e., Mg²⁺ and Mn²⁺. The K_m value for myo-inositol hexaphosphate has been found to be 0.41 × 10^?4m and V is 90.0 nmol of P_i transferred per milligram of protein per 20 min. K_m for ADP is 0.88 × 10^-4m and V is 83.3 nmol of phosphorus transferred to ADP per milligram of protein per 20 min. The ADP phosphotransferase reaction is reversible to the extent of about 50% of the forward reaction. dADP is partly effective as an acceptor but other ribonucleoside mono- and diphosphates cannot substitute for ADP. The products ATP and myo-inositol pentaphosphate have been confirmed by several criteria. It has also been shown that this enzyme transfers phosphate only from a specific phosphoryl group (C-2 position) of myo-inositol hexaphosphate for the synthesis of ATP and 1,3,4,5,6-myo-inositol pentaphosphate or pentakis (dihydrogen phosphate).     

Characterization of Mg2+-ATPase from sheep kidney medulla: magnetic resonance and kinetic studies.

Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg²⁺ or Mn²⁺ indicate a biphasic response to divalent cations. Apparent K_m values of 23 μm for free Mg²⁺ and 3.3 μm for free Mn²⁺ are obtained at low levels of added metal, while K_m values of 0.50 mm for free Mg²⁺ and 0.43 mm for free Mn²⁺ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg²⁺-ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent K_m values for free ATP of 6.0 and 1.4 μm with Mg²⁺ and Mn²⁺, respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that K_m values for free ATP of 8.0 and 2.0 mm are obtained for Mg²⁺ and Mn²⁺, respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg²⁺-ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with K_i values of 10.5 μm and 4.5 mm. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn²⁺ per 469,000 g of protein. The Mn²⁺ binding studies yield a K_D for Mn²⁺ at the single high affinity site of 2 μm, in good agreement with the kinetically determined activator constant for Mn²⁺ at low Mn²⁺ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn²⁺ per single high affinity Mn²⁺ site. The K_D for Mn²⁺ at these sites is 0.55 mm, in good agreement with the kinetic activator constant for Mn²⁺ of 0.43 mm, consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn²⁺ in the presence of the enzyme is also consistent with the tight binding of a single Mn²⁺ ion per 469,000 M_r protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn²⁺ at the single tight site, ?_b, of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn²⁺ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10^?9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg²⁺-ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg²⁺-ATPase described here are discussed.     

A comparison of kinetic parameters obtained with three major non-interconvertible isozymes of rat pyruvate kinase

The kinetic properties of partially purified kidney cortex, liver and muscle isozymes of rat pyruvate kinase (EC 2.7.1.40) were compared. The liver and kidney cortex enzymes were isolated in forms which were homotropically activated by phosphoenolpyruvate and heterotropically activated by fructose-1,6-diphosphate. In the absence of added modulators, the liver enzyme was less active, but both isozymes were fruther inactivated by l-alanine, l-phenylalanine or ATP. The liver enzyme was relatively more sensitive to ATP, but less sensitive to l-phenylalanine. The muscle enzyme, on the other hand, was isolated in a more active form which was insensitive to ATP or l-alanine inhibition and of intermediate sensitivity to l-phenylalanine inhibition. In the presence of l-phenylalanine, muscle enzyme also underwent homotropic and heterotropic activation. Not any of the isozymes were inhibited by NADH.All three isozymes were activated by K⁺ or NH₄⁺. NH₄⁺ was the more effective activator for the kidney cortex or liver enzymes, in the former case because of a greater affinity, the latter because of a higher catalytic efficiency. Of the divalent cations tested only Mg²⁺ and Mn²⁺ activated. All three isozymes had lower maximal rates when activated by Mn²⁺, but this ion also consistently acted as a typical K-type activator.Evidence also was obtained which suggested that the change from one conformational form to another might take minutes and therefore, measured kinetic parameters could reflect conformational as well as catalytic phenomena. This observation, plus suggested independent subunit interactions, were considered to be evidence favoring a sequential rather than a concerted mechanism of conformational transition.     

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme.Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α₂ An ATP analogue, γ-(p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA^Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K₁ = 1 × 10^?3m (K_mfor ATP = 2 × 10^?4m). The covalent photoaddition of azido-ATP³ results in complete loss of enzymatic activity in both the ATP-[³²P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme.Two binding sites for tRNA^Trp have been found from complex formation at pH 5.8 in the presence of Mg²⁺. The two tRNA molecules bind, with K_dis = 3.6 × 10^?8m and K_dis = 0.9 × 10^?6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA.N-chlorambucilyl-tryptophanyl-tRNA^Trp and TRSase form a complex with K_dis = 5.5 × 10^?8m at pH 5.8 in the presence of 10 mm-Mg²⁺. This value is similar to the value of K_dis for tryptophanyl-tRNA of 4.8 × 10^?8m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA^Trp whereas the tRNA devoid of tRNA^Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected.Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [³²P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA^Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[³²P]pyrophosphate exchange and aminoacylation activity.The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein.Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.     

Sulfur assimilation in c(4) plants: intercellular compartmentation of adenosine 5'-triphosphate sulfurylase in crabgrass leaves

The activity of adenosine 5′ triphosphate sulfurylase was determined in crabgrass mesophyll cells, bundle sheath strands, and whole leaf extracts. The enzyme was assayed by following molybdate-dependent pyrophosphate release from ATP, ³⁵SO₄²⁻ incorporation into adenosine 5′ phosphosulfate, and ATP synthesis dependent upon adenosine 5′ phosphosulfate and inorganic pyrophosphate. With all assays, greater than 90% of the activity was found in extracts from bundle sheath strands. The activities in whole leaf extracts were consistently intermediate between the activities of mesophyll and bundle sheath extracts and extract-mixing experiments gave no indication of enzyme activation or inhibition in vitro. Whole leaf activities were several hundred-fold less than concurrent measurements of ribulose 1,5-bisphosphate and phosphoenolpyruvate carboxylase activities, which is interpreted as being consistent with the relative amounts of elemental carbon and sulfur found in higher plants. A hypothesis is presented for the intercellular compartmentation of sulfur assimilation in relationship to NO₃⁻ and CO₂ assimilation in leaves of C₄ plants.