Regulation of Crassula argentea phosphoenolpyruvate carboxylase in relation to temperature.

The effect of temperature on the kinetic parameters of phosphoenolpyruvate carboxylase purified from Crassula argentea was such that both the Vmax and Km(MgPEP) values tended upward over the range from 11 to 35 degrees C. The increased rate at low temperatures due to the low Km is at least partially offset by the increased Vmax at higher temperatures, potentially leading to a broad plateau of enzyme activity and a relatively small effect of temperature on the enzyme. The cooperativity was negative at 11 degrees C, but above 15 degrees C it became positive. The presence of 5 mM glucose-6-phosphate has relatively little effect on Vmax but it clearly reduces Km and overcomes any effect of temperature on this parameter in the range studied. Positive cooperativity is observed only at temperatures above 25 degrees C. The size of the native enzyme, as determined by dynamic light scattering, was strongly toward the tetrameric form. At a temperature of 40 degrees C and above, a considerable oligomerization takes place. No loss of activity can be observed in this range of temperature. In the presence of either glucose-6-phosphate or magnesium phosphoenolpyruvate, at temperatures under 25 degrees C, the equilibrium is displaced toward higher levels of aggregation. Maximal accumulation of lead malate occurred at 10 to 12 degrees C in vivo with reduction to about 25% at 35 degrees C. Glucose-6-phosphate followed a similar curve in response to temperature, but the overall difference was about 50%. The sum of phosphoenolpyruvate plus pyruvate is level at night temperatures below 25 degrees C, doubling at 35 degrees C. Calculated concentrations of malate, glucose-6-phosphate, and phosphoenolpyruvate plus pyruvate indicate that the concentrations present are equal to or greater than Ki, Ka, and Km values for these metabolites, respectively.     

The influence of pH on substrate form specificity of phosphoenolpyruvate carboxylase purified from Crassula argentea

Purified phosphoenolpyruvate carboxylase from both the crassulacean acid metabolism plant Crassula argentea and the C4 plant Zea mays was shown by kinetic studies at saturating fixed-varying concentrations of free mg2+ to selectively use the metal-complexed form of phosphoenolpyruvate when assayed at pH 8.0. A similar response to added magnesium at high free phosphoenolpyruvate concentrations was obtained for both enzymes, consistent with the use of the complex as the substrate. Kinetic studies at pH 7.0 indicated that at this pH the total concentration of phosphoenolpyruvate (including both free and metal-complexed forms) could be used by the enzyme from C.argentea while the C4 enzyme still utilized the complex. The loss of specificity induced by the decrease in the pH of the assay medium was accompanied by a decrease in the Km of this enzyme for phosphoenolpyruvate whatever the form considered and an increase in Vmax/Km. In contrast, a similar decrease of pH led to an increased Km of the C4 enzyme for phosphoenolpyruvate and a decrease of Vmax/Km. For the enzyme from C. argentea (previously shown to contain an essential arginine at the active site), protection of activity by the different forms of substrate against inactivation by the specific arginyl reagent 2,3-butanedione changes markedly with pH. At pH 8.1, the metal complex is the better protector while at pH 7.0 free phosphoenolpyruvate gives the best protection consistent with the observed kinetic changes in substrate form utilization. The relationship between the enzyme affinity for substrate, substrate specificity, and the requirement for magnesium for substrate turnover is discussed.     

Identification of substrate and effector binding sites of phosphoenolpyruvate carboxylase from Crassula argentea. A possible role of phosphoenolpyruvate as substrate and activator

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) purified from leaves of the crassulacean acid metabolism plant (Crassula argentea) was chemically modified by the specific arginyl reagent 2,3-butanedione. Modification resulted in enzyme inactivation which followed pseudo first-order kinetics. Participation of arginyl residues involved in the binding of or response to both phosphoenolpyruvate and malate, respectively, was established. Inactivation and protection studies suggest the presence of three sites involved in the binding of the substrate, phosphoenolpyruvate, the activator, glucose 6-phosphate, and the inhibitor, malate. Studies using both fluorescence measurements of binding and steady-state kinetic methods indicate that phosphoenolpyruvate can bind both to the active site and to the activator site. Evidence for stimulation of the activity of phosphoenolpyruvate carboxylase upon the binding of substrate to the activation site was provided by kinetic studies using AMP, previously shown to be a specific ligand for the activation site.     

Regulation of phosphoenolpyruvate carboxylase from Crassula by interconversion of oligomeric forms

Using size-exclusion high-performance liquid chromatography, it is shown that phosphoenolpyruvate carboxylase from Crassula argentea, a crassulacean acid metabolism (CAM) plant, exists primarily in the form of a tetramer of a 100-kDa subunit at night and as a dimer of the same subunit during the day. The tetrameric enzyme from night leaves is not inhibited by malate, while the dimeric form from day leaves can be completely inhibited by malate. The purified day, or dimer, form of the enzyme can be converted to the tetramer by concentration and exposure to Mg2+. When thus converted, the tetramer is insensitive to malate inhibition, and is more strongly activated by glucose 6-phosphate than the dimer. The purified night, or tetramer, form is converted to the dimer by incubation for 60 min at pH 8.2. This enzyme may also be converted to the dimer by adding 1.5 mM malate to the elution buffer, but preincubation for 15 min with phosphoenolpyruvate prevents disaggregation when chromatographed with buffer containing malate. Preincubation with 1mM EDTA and subsequent chromatography with buffer containing malate shows a progressive dissociation of the tetrameric form with increasing time of preincubation. The implications of these observations for the diurnal regulation of phosphoenolpyruvate carboxylase in CAM metabolism are discussed.     

A kinetic investigation of phosphoenolpyruvate carboxylase from Zea mays.

The reaction catalyzed by phosphoenolpyruvate carboxylase from Zea mays has been studied kinetically. Results of initial velocity patterns and inhibition studies indicate that phosphoenolpyruvate carboxylase has a random sequential mechanism in which there is a high level of synergism in the binding of substrates. The preferred order of addition of reactants is Mg2+, phosphoenolpyruvate, and bicarbonate. The binding of Mg2+ is at equilibrium. Values for the various kinetic parameters are KiMg = 2.3 +/- 0.4 mM, KPEP = 3.6 +/- 0.6 mM, KiPEP = 0.2 +/- 0.07 mM, and Kbicarbonate = 0.18 +/- 0.04 mM. In addition, double inhibition experiments have been performed to examine the nature of the active site interactions with the putative intermediates, carboxy phosphate and the enolate of pyruvate. Highly synergistic inhibition of phosphoenolpyruvate carboxylase was observed in the presence of oxalate and carbamyl phosphate (alpha = 0.0013). However, an antisynergistic relationship exists between oxalate and phosphonoformate (alpha = 2.75).     

Effect of Diethylpyrocarbonate on the Allosteric Properties of Phosphoenolpyruvate Carboxylase from Crassula argentea

Phosphoenolpyruvate carboxylase from the Crassulacean acid metabolism plant Crassula argentea was substantially desensitized to the effects of regulatory ligands by treatment with diethylpyrocarbonate, a reagent which selectively modifies histidyl residues. Desensitization of the enzyme to the inhibitor malate and the activator glucose 6-phosphate was accompanied by the appearance of a peak in the ultraviolet difference spectrum at 240 nanometers, indicating the formation of ethoxyformylhistidyl derivatives. Hydroxylamine reversed part of the spectral change under native conditions, and almost all of the change under denaturing conditions, but failed to restore sensitivity to effectors. The pH profiles of desensitization to malate and glucose 6-phosphate indicated the involvement of groups on the enzyme with pK, values of 6.8 and 6.4, respectively. Under denaturing conditions, a total of 15 histidine residues per subunit were modified by diethylpyrocarbonate, whereas for the native enzyme nine histidines were modified per subunit. Effector desensitization occurs after the modification of two to three histidyl residues per subunit. The presence of malate reduced the apparent rate constant for desensitization by 60%, suggesting that the modification occurred at the malate binding site. Diethylpyrocarbonate treatment also eliminated the kinetic lag caused by malate. Glucose 6-phosphate did not protect the enzyme against diethylpyrocarbonate-induced desensitization.     

A comparison of the kinetic properties of phosphoenolpyruvate carboxylase from guard-cell and mesophyll-cell protoplasts of Commelina communis

Some kinetic properties of partially purified phosphoenolpyruvate carboxylase (PEPCase) from guard-cell and mesophyll-cell protoplasts of Commelina communis are described. The PEPCase activity inherent to each cell type was determined and the apparent K _m (phosphoenolpyruvate) and K _i (malate) were compared. Malate sensitivity was much higher (K _i malate 0.4 mol m^–3) in the extract of guard-cell protoplasts than in that of mesophyllcell protoplasts (K _i malate 4.2 mol m^–3). The stimulation of activity by glucose-6-phosphate in the presence of malate (deinhibition) was also investigated in extracts from both cell types and was found to be similar to previously reported results with epidermal tissue. The effect of contamination of an extract of guard-cell protoplasts with mesophyll-cell protoplasts was measured in the presence and absence of malate. It was found that a small amount to mesophyll-cell contaminant appears to desensitize the malate inhibition of PEPCase from guard-cell protoplasts. It is concluded that experiments which use epidermal tissue to study guardcell PEPCase may give misleading information as a consequence of mesophyll contamination.Abbreviations Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase     

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Effects of phosphate on the activity, stability and regulatory properties of phosphoenolpyruvate carboxylase from the C4 plant Cynodon dactylon

The extraction of phosphoenolpyruvate carboxylase, PEPC (EC 4.1.1.31) from leaves of Cynodon dactylon (L.) Pers. with phosphate buffer (pH 7.4, 105 mM) was advantageous in comparison to the usual extraction with Tris-HCl buffer (pH 7.4, 100 mM); a higher activity was obtained, which was most evident at low substrate (phosphoenolpyruvate) concentrations. The PEPC activity was stable under dilution or in storage for at least 48 h at room temperature. The effects of phosphate buffer were not due to inhibition of phosphatase(s) action during the extraction, since they were also observed when the phosphates were added after the extraction with Tris-HCl. The phosphate-extracted enzyme was less responsive to both L-malate inhibition and activation by glucose-6-phosphate. The effects of phosphates might be due to preferential exclusion from the enzymic protein domain and, therefore, to a confinement of the enzyme to a fraction of the total volume. This revised version was published online in August 2006 with corrections to the Cover Date.     

Regulation of Phosphoenolpyruvate Carboxylase from Crassula argentea: Further Evidence on the Dimer-Tetramer Interconversion

Phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants during the day exists in dimeric form the activity of which is strongly inhibited by malate. Enzyme purified from Crassula leaves collected during the day and stored at −70°C for 49 days shows a steady progression of change from dimer to tetramer, and this change in oligomeric state is accompanied by a decrease in the sensitivity of the enzyme to inhibition by malate. At 10 minutes preincubation of enzyme after 11 days storage—which is composed of an equilibrium mixture of dimer and tetramer—with malate causes most of the enzyme to be converted to dimer and increases the sensitivity of the enzyme to malate inhibition during assay. Preincubation with phosphoenolpyruvate shifts the equilibrium toward the tetrameric form and reduces the maximal inhibition produced by 5 millimolar malate to less than 20%. However, none of the treatments used resulted in shifting the oligomerization equilibrium completely in either direction. Thus the question of whether some covalent modification of the enzyme, such as phosphorylation, is required to permit complete changes in equilibrium remains open.     

A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity

The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V_max with no apparent effect on K_m. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.     

燕子掌挥发油化学成分的研究

应用气象色谱-质谱联用技术对燕子掌挥发油化学成分进行了分析研究,共鉴定出66种组分与燕子掌主要挥发性化学成分以苯乙醇、2,6,6-三甲基-2,4-环庚二烯-1-酮、6,10,14-三甲基十五烷-2-酮、十六烷酸甲酯、十六烷酸乙酯、十八烷酸甲酯为主要成分,化合物类型以酮、酯、类等化合物为主,其中十六烷酸甲酯的含量最高,占挥发油总量的26．13％。     

The dimeric form of phosphoenolpyruvate carboxylase isolated from maize: physical and kinetic properties

Phosphoenolpyruvate carboxylase purified from maize was a homodimer of molecular weight 200 kDa and was readily converted to a tetrameric form in the presence of Mg2+ plus PEP or Mg2+ alone. During the assay, the enzyme activity increased with time, reaching a steady state after a discernible lag, suggesting its hysteretic nature. The hystereses was not due to oligomerization of the enzyme as the lag time tau was independent of the enzyme concentration and the lag was not abolished on preincubation with 25 mM Mg2+, the condition under which the enzyme existed in tetrameric form. Nevertheless, the lag could be abolished on preincubating the enzyme with PEP plus Mg2+, indicating that the hystereses is due to a PEP plus Mg2(+)-induced slow transition of the enzyme to an activated state during the catalysis. During steady state, the enzyme showed cooperative kinetics for PEP and Mg2+ at pH 7. It had two binding sites with nearly 10-fold difference in affinities for PEP and Mg2+.     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

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.     

Physical and kinetic properties of photosynthetic phosphoenolpyruvate carboxylase in developing apple fruit

Phosphoenolpyruvate carboxylase (PEPC) was partially purified from young developing apple fruit, cultivars Golden Delicious and Cox's Orange Pippin. Freeze-drying of tissue reduced the yield of PEPC activity compared to samples stored at 4°. Activities measured by H¹⁴CO₃⁻ incorporation exceeded the spectrophotometric assay for the enzyme with coupled NADH-malate dehydrogenase (MDH) by up to 60%. The enzyme could be stored at −16° with glycerol and bovine serum albumin for several months without loss of activity. Thermal inactivation of PEPC occurred after heating to 75° for 3 min when MDH was still slightly active. Inhibition of PEPC activity by endogenous phenolics could be prevented by grinding in liquid nitrogen in the presence of polyvinylpyrrolidine and dithiothreitol. Apparent K_m (PEP) and V_max values compared more favourably with those obtained from a C₃-species (spinach) than from a C₄-species (maize). l-Malate (5 mM) inhibited fruit PEPC by 22%; this was decreased to 12% by addition of glucose-6-phosphate (2 mM). From kinetic and effector experiments PEPC in the apple fruit is concluded to be a non-C₄ photosynthetic enzyme.     

Involvement of thiol groups in the activity of phosphoenolpyruvate carboxylase from maize leaves

Purified maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) was completely inactivated by several thiol-modifying reagents, including, CuCl₂, CdCl₂ and N-ethylmaleimide. The inactivation by CuCl₂ could be reversed by dithiothreitol, suggesting the involvement of vicinal dithiols in the inactivation process.Complete inactivation of phosphoenolpyruvate carboxylase was correlated with the incorporation of two mol (³H)N-ethylmaleimide per 100-kilodalton subunit. The total protection of the enzyme against N-ethylmaleimide inactivation afforded by the substrate, phosphoenolpyruvate, was correlated with the protection of one mol (³H)N-ethylmaleimide reactive residue per mol subunit.The complete inactivation of phosphoenolpyruvate carboxylase by N-ethylmaleimide and the protection afforded by phosphoenolpyruvate against modification suggest the presence of an essential cysteine residue in the catalytic site of the C₄ leaf enzyme.Abbreviations PEP, phosphoenolpyruvate - Mops, 4-morpholinepropanesulphonic acid (Consejo Nacional de Investigaciones Científicas y Técnicas, Fundación M. Lillo y U.N. de Rosario).     

A steady-state kinetic study on the catalytic mechanism of ribulose bisphosphate carboxylase from soybean

The catalytic mechanism of soybean ribulose bisphosphate carboxylase was examined, through a study of the steady-state kinetic behavior of the fully activated enzyme using short time assays. The effects of substrates, products, alternative products, and two dead-end inhibitors were investigated. High concentrations of both substrates were observed to lead to nonhyperbolic relationships: concentrations of bicarbonate greater than 15 mm inhibited and concentrations of ribulose bisphosphate greater than 0.2–0.5 mm stimulated enzyme activity over that expected from a hyperbolic fit to the data. The kinetic patterns obtained and the nonhyperbolic behavior of substrates are interpreted to suggest that the binding of substrates and the release of products follow a steady-state random mechanism. The substrate activation by ribulose bisphosphate is likely to be physiologically significant.     

Escherichia coli phosphoenolpyruvate carboxylase: effect of allosteric inhibitors on the kinetic parameters and sedimentation behavior

Various metabolic intermediates and chemically related compounds were studied to determine their effects on the catalytic activity and sedimentation behavior in sucrose gradients of phosphoenolpyruvate carboxylase of Escherichia coli. Two types of enzyme-inhibitor interactions were found.In the first type of inhibition, the inhibitor was functionally competitive with acetyl coenzyme A and had the ability to stabilize the tetrameric form of the enzyme, as evidenced by its effect on the apparent sedimentation rate of the enzyme in sucrose gradients. These compounds were approximately equivalent in length to four-carbon dicarboxylic acids and had to be ionized with negative charges at both ends. In addition, the strong inhibitors (aspartate, malate, fumarate, cysteine, and tartrate) have an electron-attracting group as an L-α substituent. Fumarate was also a strong inhibitor, and it too contained an electron-attracting group in the form of an olefinic bond.The second type of inhibition was exhibited uniquely by maleate. Maleate inhibited the enzyme strongly, but it was not competitive with acetyl coenzyme A nor could it stabilize the tetrameric form of the enzyme at concentrations that were highly inhibitory. The pattern of inhibition by maleate could be converted to that seen with the other inhibitors in the presence of aspartate. This indicates that each class of inhibitor is competitive with the other and that all allosteric inhibitors may share the same binding site.