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1.
Wu MX  Wedding RT 《Plant physiology》1992,100(1):382-387
Phosphoenolpyruvate carboxylase (PEPC) purified from maize (Zea mays L.) leaves associates with maize leaf chloroplast membrane in vitro. The binding of PEPC to the membrane results in enzyme inactivation. A protein isolated from a maize leaf chloroplast membrane preparation inactivated PEPC. Treatment with membrane preparation or with partially purified inactivating protein accelerates PEPC inactivation at low temperature (4°C). Interaction of PEPC with chloroplast membrane or inactivating protein may inactivate the enzyme by influencing dissociation of the enzyme active tetramer.  相似文献   

2.
Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg2+ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.  相似文献   

3.
Diethyl oxaloacetate was found to be a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase activity with respect to the substrate phosphoenolpyruvate. The Ki values, based on total diethyl oxaloacetate, decreased with increasing pH, while the Ki values, based on the enol tautomer (average of 4 M), were similar and independent of pH. The results suggest that inhibition is dependent on the enol tautomer. Diethyl oxaloacetate was a weak inhibitor following treatment of the enzyme with dithiothreitol; inhibition could be restored by treatment with diamide, indicating inhibition depends on the reduction state of thiol groups on the enzyme.Abbreviations DTT dithiothreitol - HPLC high performance liquid chromatography - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine  相似文献   

4.
The phosphoenolpyruvate carboxylase from maize leaf was strongly inhibited by 2-phosphoglycollate. The pH of the reaction did not influence the extent of inhibition by 2-phosphoglycollate. The kinetic analysis of the inhibition data by Lineweaver-Burk method showed that 2-phosphoglycollate inhibition was competitive with respect to phosphoenolpyruvate. The secondary plot of the data showed nonlinearity indicating that there may be two 2-phosphoglycollate binding sites with Ki values of 0.4 mM and 0.16 mM. The biphasic nature of the inhibition was also evident when the data were plotted using the method of Dixon. 2-phosphoglycollate inhibition was uncompetitive with respect to Mg2+ suggestting that it binds only to enzyme-Mg2+ complex.  相似文献   

5.
Light modulation of maize leaf phosphoenolpyruvate carboxylase   总被引:1,自引:3,他引:1       下载免费PDF全文
Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH4)2SO4 fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition.  相似文献   

6.
Maize phosphoenolpyruvate carboxylase (PEPC) was rapidly and completely inactivated by very low concentrations of trypsin at 37 degrees C. PEP+Mg2+ and several other effectors of PEP carboxylase offered substantial protection against trypsin inactivation. Inactivation resulted from a fairly specific cleavage of 20 kDa peptide from the enzyme subunit. Limited proteolysis under catalytic condition (in presence of PEP, Mg2+ and HCO3) although yielded a truncated subunit of 90 kDa, did not affect the catalytic function appreciably but desensitized the enzyme to the effectors like glucose-6-phosphate glycine and malate. However, under non-catalytic condition, only malate sensitivity was appreciably affected. Significant protection of the enzyme activity against trypsin during catalytic phase could be either due to a conformational change induced on substrate binding. Several lines of evidence indicate that the inactivation caused by a cleavage at a highly conserved C-terminal end of the subunit.  相似文献   

7.
Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg2+ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.  相似文献   

8.
The aim of this work was to investigate how light regulates the activity of phosphoenolpyruvate carboxylase in vivo in C4 plants. The properties of phosphoenolpyruvate carboxylase were investigated in extracts which were rapidly prepared (in less than 30 seconds) from darkened and illuminated leaves of Zea mays. Illumination resulted in a significant decrease in the S0.5(phosphoenolpyruvate) but there was no change in Vmax. The form of the enzyme from illuminated leaves was less sensitive to malate inhibition than was the form from darkened leaves. At low concentrations of phosphoenolpyruvate, the activity of the enzyme was strongly stimulated by glucose-6-phosphate, fructose-6-phosphate, triose-phosphate, alanine, serine, and glycine and was inhibited by organic acids. The enzyme was assayed in mixtures of metabolites at concentrations believed to be present in the mesophyll cytosol in the light and in the dark. It displayed low activity in a simulated `dark' cytosol and high activity in a simulated `light' cytosol, but activities were different for the enzyme from darkened compared to illuminated leaves.  相似文献   

9.
The hysteretic nature of phosphoenolpyruvate carboxylase from maize was investigated at pH 7 by (a) transient kinetic studies, (b) kinetics of inhibition by 2-PG, a structural analog of PEP, and (c) effect of 2-PG on equilibrium binding of Mg2+. The lag time as a function of substrate concentration was nonlinear with an oblique asymptote. During steady state, cooperative kinetics for Mg2+ was changed to hyperbolic kinetics in the presence of 2-PG. Studies on the equilibrium binding of Mg2+ with the help of an external fluorescent probe, 8-anilino-6-naphthalinosulfonate showed that the hyperbolic binding of Mg2+ was changed to cooperative binding in the presence of 2-PG. On the basis of these results along with the results presented in the preceding paper, a fully concerted sequential model with subunit interaction is proposed for PEPC.  相似文献   

10.
In vitro phosphorylation of maize leaf phosphoenolpyruvate carboxylase   总被引:3,自引:2,他引:1  
Budde RJ  Chollet R 《Plant physiology》1986,82(4):1107-1114
Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with 32P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ-32P]ATP or [β-32P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acid-stable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase from illuminated maize leaves by an endogenous protein kinase resulted in a partial inactivation (~25%) of the enzyme when assayed at pH 7 and subsaturating levels of PEP, effector modulation by l-malate and glucose-6-phosphate was relatively unaffected. Changes in the aggregation state of maize PEPCase (homotetrameric native structure) were studied by nondenaturing electrophoresis and immunoblotting. Enzyme from leaves of illuminated plants dissociated upon dilution, whereas the protein from darkened tissue did not dissociate, thus indicating a physical difference between the enzyme from light- versus dark-adapted maize plants.  相似文献   

11.
12.
Phosphoenolpyruvate carboxylase has been purified to homogeneity from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. The ratio of specific activities in crude extracts and the purified enzyme suggests that the enzyme is a major soluble protein in the tissue. The enzyme has a sedimentation coefficient (s20,w) of 12.3S and a molecular weight, determined by sedimentation equilibrium, of 400,000 daltons. Dissociation of the enzyme and electrophoresis on dodecyl sulfate polyacrylamide gels yields a single stained band which corresponds to a subunit weight of 99,000 daltons. Thus it appears that the native enzyme is composed of four identical or similar polypeptide chains.  相似文献   

13.
Effects of pH on inactivation of maize phosphoenolpyruvate carboxylase   总被引:1,自引:0,他引:1  
Maize leaf phosphoenolpyruvate carboxylase (PEPC) is inactivated by incubation at pH's above neutrality. Both the amount and the rapidity of inactivation increase as the pH rises. The presence of phosphoenolpyruvate (PEP), malate, glucose 6-phosphate and dithiothreitol in the incubation medium give protection to the enzyme. While the presence of PEP during incubation at pH 8 prevents inactivation, the level of PEP in the assay after incubation has no effect on the relative inactivation. When the enzyme is incubated at pH 7 with 5 mM malate (a treatment known to cause dimerization) subsequent assay at saturating levels of MgPEP completely restores activity while assay at less than Km MgPEP produces greater than 99% inhibition of the same sample, showing that high PEP concentration has reconverted the PEPC to the malate-resistant tetramer. Thus the protective effect of PEP against inactivation at high pH probably is not related to its effect on the aggregation state of the enzyme but rather is due to the presence of PEP at the active site. Protection of PEPC at pH 8 by EDTA and its inactivation by low concentrations of Cu2- indicates that the loss of activity at high pH probably is in a sense an artifact resulting from the binding to a deprotinated cysteine of heavy metal ions contaminating the enzyme preparation or present in reagents. This suggests that caution should be used in the interpretation of experiments involving PEPC activity at alkaline pH's.  相似文献   

14.
Nitrate and alanine were found to stimulate partially purified maize leaf phosphoenolpyruvate carboxylase under specific assay conditions. Both metabolites stimulated the enzyme at low pH (7.0-7.5) and low substrate levels (1mM phosphoenolpyruvate). Nitrate was found to have a biphasic effect on the enzyme, stimulating at low concentrations (1mM-3mM), with a decrease in stimulation at higher levels. Nitrate caused inhibition of activity at pH 8.0 and although alanine caused some stimulation in activity at pH 8.0 this was not as marked as at the lower pH levels.  相似文献   

15.
Incubation of purified phosphoenolpyruvate carboxylase from Zea mays L. leaves with dithiothreitol resulted in an almost 2-fold increase in the enzymic activity. The activated enzyme showed the same affinity for its substrates and the same sensitivity with respect to malate and oxalacetate inhibition. The activation induced by dithiothreitol was reversed by diamide, an oxidant of vicinal dithiols, suggesting that the redox state of disulfide bonds of the enzyme may be important in the expression of the maximal catalytic activity.

Titration of thiol groups before and after activation of maize phosphoenolpyruvate carboxylase by dithiothreitol shows an increase of the accessible groups from 8 to 12 suggesting that the reduction of two disulfide bonds accompanied the activation. The thiols exposed by the treatment with dithiothreitol were available to reagents in nondenatured enzyme and two of them were reoxidized to a disulfide bond by diamide. It is concluded that the mechanism of phosphoenolpyruvate carboxylase activation by dithiothreitol involves the net reduction of two disulfide bonds in the enzyme.

  相似文献   

16.
It has been a common practice to assay phosphoenolpyruvate carboxylase (PEPC) under high, nonphysiological concentrations of Mg(2+) and bicarbonate. We have performed kinetic studies on the enzyme from maize (Zea mays) leaves at near physiological levels of free Mg(2+) (0.4 mM) and bicarbonate (0.1 mM), and found that both the nonphosphorylated and phosphorylated enzymes exhibited a high degree of cooperativity in the binding of phosphoenolpyruvate, a much lower affinity for this substrate and for activators, and a greater affinity for malate than at high concentrations of these ions. Inhibition of the phosphorylated enzyme by malate was overcome by glycine or alanine but not by glucose-6-phosphate, either in the absence or presence of high concentrations of glycerol, a compatible solute. Alanine caused significant activation at physiological concentrations, suggesting a pivotal role for this amino acid in regulating maize leaf PEPC activity. Our results showed that the maximum enzyme activity attainable in vivo would be less than 50% of that attainable in vitro under optimum conditions. Therefore, the high levels of PEPC protein in the cytosol of C(4) mesophyll cells might be an adaptation for sustaining the steady-state rate of flux through the photosynthetic CO(2) assimilation pathway despite the limitations imposed by the PEPC kinetic properties and the conditions of its environment.  相似文献   

17.
An antibody for phosphoenolpyruvate carboxylase was used to isolate and to quantitate the enzyme from greening maize (cv. KOU 6) leaves. The increase in enzyme activity during greening was due to de novo synthesis, which was paralleled by increases in enzyme protein and incorporation of leucine. The light-induced activity was due to one specific isoenzyme. The action spectrum for enzyme synthesis had red and blue peaks.  相似文献   

18.
19.
Phosphoenolpyruvate carboxylase (PEPCase) from light- and dark-adapted maize leaves was rapidly purified in the presence of L-malate and glycerol to apparent electrophoretic homogeneity by ammonium sulfate fractionation, hydroxylapatite chromatography, and fast-protein liquid chromatography on Mono Q. The resulting preparations were totally devoid of pyruvate, orthophosphate dikinase protein based on immunoblot analysis. Throughout the purification, both forms of PEPCase retained their different enzymatic properties. The specific activity of the light enzyme was consistently about twice that of the dark form when assayed at suboptimal (but physiological) pH (pH 7.0-7.3), and the former was also less sensitive to feedback inhibition by L-malate than that from darkened leaves under various conditions. Covalently bound phosphate and high-performance liquid chromatography-based phosphoamino acid analyses showed that both forms of purified PEPCase were phosphorylated exclusively on serine residues, but the degree of phosphorylation was about 50% greater in the light enzyme. Notably, incubation of purified PEPCase in vitro with exogenous alkaline phosphatase led to an increase in malate sensitivity and a decrease in specific activity of the light form enzyme to levels observed with the dark form, which was essentially not affected by phosphatase treatment. These results with the purified enzyme from light- and dark-adapted maize leaves indicate that the light-induced changes in activity and malate sensitivity of C4 PEPCase are related, at least in part, to the degree of covalent seryl phosphorylation of the protein in vivo.  相似文献   

20.
The analogue (Z)-phosphoenol-3-fluoropyruvate [(Z)-3-fluoro-2-(phosphono-oxy)propenoic acid] was tested as substrate of maize leaf phosphoenolpyruvate carboxylase. Studies with NaH14CO3 indicate that the analogue is carboxylated by the enzyme. However, this reaction accounts for only one-tenth of the activity measured by Pi liberation. The rest of the analogue is merely dephosphorylated. This is the first analogue for which both carboxylation and dephosphorylation have been observed.  相似文献   

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