Inhibition of phosphoenolpyruvate carboxylase from C4 plants by malate and aspartate

1975 ◽  
Vol 53 (17) ◽  
pp. 1925-1933 ◽  
Author(s):  
S. C. Huber ◽  
G. E. Edwards
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Maximum Velocity ◽  
C4 Plants ◽  
Digitaria Sanguinalis ◽  
Leaf Extracts ◽  
Ph Optimum ◽  
Pep Carboxylase ◽  
Assay Mixture ◽  
Acid Inhibitor ◽  
Rate Limiting

The activity of phosphoenolpyruvate carboxylase in leaf extracts of C4 plants has been shown to be inhibited by the C4 acids malate and aspartate. The magnitude of inhibition observed with phosphoenolpyruvate carboxylase from Digitaria sanguinalis leaves was dependent on the pH of the assay mixture and the concentration of phosphoenolpyruvate (PEP), Mg2+. and the C4 acid inhibitor. The percentage inhibition decreased with increasing pH up to 8.5. At rate-limiting concentrations of phosphoenolpyruvate the pH optimum of the enzyme was 7.8, whereas the enzyme partially inhibited by malate or aspartate had a pH optimum of roughly 8.3. Malate and aspartate inhibited the enzyme by increasing the apparent Km for phosphoenolpyruvate without altering the maximum velocity, such that inhibition was greatest at rate-limiting concentrations of PEP but was not observed at saturating (3 mM) substrate levels. Glucose-6-P. an activator of PEP carboxylase, decreased the apparent Km for PEP and reversed the inhibition by malate and aspartate. Inhibition was also dependent on the concentration of Mg2+. At 1 mM Mg2+, 1 mM malate caused 60% inhibition, but at 5 mM Mg2+ the inhibition was reduced to 15%. Mg2+ at concentrations from 1 to 5 mM had no effect on the apparent Km for PEP. With Sephadex extracts of PEP carboxylase from leaves of D. sanguinalis, malate and aspartate were equally inhibitory, with roughly 2 mM levels required for 50% inhibition. Both malate and aspartate were inhibitory with PEP carboxylase extracted from leaves of a variety of C4∙ but not C3∙ plants. The significance of the observed inhibition and activation of PEP carboxylase by metabolites is discussed in relation to C4 photosynthesis.

scholarly journals Properties and regulation of leaf nicotinamide–adenine dinucleotide phosphate–malate dehydrogenase and ‘malic’ enzyme in plants with the C4-dicarboxylic acid pathway of photosynthesis

1970 ◽  
Vol 119 (2) ◽  
pp. 273-280 ◽  
Author(s):  
Hilary S. Johnson ◽  
M. D. Hatch
Keyword(s):  
Dicarboxylic Acid ◽  
Malate Dehydrogenase ◽  
Malic Enzyme ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Maximum Velocity ◽  
Enzyme Reaction ◽  
Reverse Direction ◽  
Leaf Extracts ◽  
Ph Optimum ◽  
Acid Pathway

1. NADP–malate dehydrogenase and `malic' enzyme in maize leaf extracts were separated from NAD–malate dehydrogenase and their properties were examined. 2. The NADP–malate dehydrogenase was nicotinamide nucleotide-specific but otherwise catalysed a reaction comparable with that with the NAD-specific enzyme. By contrast with the latter enzyme, a thiol was absolutely essential for maintaining the activity of the NADP–malate dehydrogenase, and the initial velocity in the direction of malate formation, relative to the reverse direction, was faster. 3. For the `malic' enzyme reaction the Km for malate was dependent on pH and the pH optimum varied with the malate concentration. At their respective optimum concentrations the maximum velocity for this enzyme was higher with Mg2+ than with Mn2+. 4. The NADP–malate dehydrogenase in green leaves was rapidly inactivated in the dark and was reactivated when plants were illuminated. Reactivation of the enzyme extracted from darkened leaves was achieved simply by adding a thiol compound. 5. The activity of both enzymes was low in etiolated leaves of maize plants grown in the dark but increased 10–20-fold, together with chlorophyll, when leaves were illuminated. 6. The activity of these enzymes in different species with the C4-dicarboxylic acid pathway was compared and their possible role in photosynthesis was considered.

CARBON DIOXIDE FIXATION AND PHOSPHOENOLPYRUVATE CARBOXYLASE IN FERROBACILLUS FERROOXIDANS

1967 ◽  
Vol 13 (11) ◽  
pp. 1413-1419 ◽  
Author(s):  
George A. Din ◽  
Isamu Suzuki ◽  
Howard Lees
Keyword(s):  
Carbon Dioxide ◽  
Phosphoenolpyruvate Carboxylase ◽  
Metabolic Regulation ◽  
Carbon Dioxide Fixation ◽  
Acidic Ph ◽  
Ph Optimum ◽  
Carboxylase Activity ◽  
Intact Cells ◽  
Pep Carboxylase

Carbon dioxide fixation was studied in intact cells and cell-free extracts of Ferrobacillus ferrooxidans. The major pathways of fixation were found to be the carboxydismutase system and the phosphoenolpyruvate (PEP) carboxylase system. PEP carboxylase activity was shown to be under metabolic regulation, similar to the regulation established in heterotrophic microorganisms.Acetyl-CoA stimulated PEP carboxylase activity, while aspartate was inhibitory. The F. ferrooxidans enzyme appeared to have a neutral or acidic pH optimum, in contrast to the same enzyme isolated from heterotrophs.

Possible involvement of phosphoenolpyruvate carboxylase and NAD-malic enzyme in response to drought stress. A case study: a succulent nature of the C4-NAD-ME type desert plant, Salsola lanata (Chenopodiaceae)

2017 ◽  
Vol 44 (12) ◽  
pp. 1219
Author(s):  
Zhibin Wen ◽  
Mingli Zhang
Keyword(s):  
Enzyme Activity ◽  
Drought Stress ◽  
Phosphoenolpyruvate Carboxylase ◽  
Malic Enzyme ◽  
C4 Plants ◽  
Leaf Water Content ◽  
Transcriptional Level ◽  
Desert Plant ◽  
Severe Stress ◽  
Real Time Quantitative Pcr

The co-ordination between the primary carboxylating enzyme phosphoenolpyruvate carboxylase (PEPC) and the further decarboxylating enzymes is crucial to the efficiency of the CO2-concentrating mechanism in C4 plants, and investigations on more types of C4 plants are needed to fully understand their adaptation mechanisms. In this study we investigated the effect of drought on carboxylating enzyme PEPC, and the further decarboxylating NAD-malic enzyme (NAD-ME) of Salsola lanata Pall. (Chenopodiaceae) – an annual succulent C4-NAD-ME subtype desert plant. We investigated enzyme activity at the transcriptional level with real-time quantitative PCR and at the translational level by immunochemical methods, and compared S. lanata with other forms of studied C4 plants under drought stress. Results showed that only severe stress limited PEPC enzyme activity (at pH 8.0) of S. lanata significantly. Considering that PEPC enzyme activity (at pH 8.0) was not significantly affected by phosphorylation, the decrease of PEPC enzyme activity (at pH 8.0) of S. lanata under severe stress may be related with decreased PEPC mRNA. The suggestion of increased phosphorylation of the PEPC enzyme in plants under moderate stress was supported by the ratio of PEPC enzyme activity at pH 7.3/8.0, as PEPC enzyme is inhibited by L-malate and the evidence of the 50% inhibiting concentration of L-malate. NAD-ME activity decreased significantly under moderate and severe stress, and coincided with a change of leaf water content rather than the amount of α-NAD-ME mRNA and protein. Leaf dehydration may cause the decrease of NAD-ME activity under water stress. Compared with other C4 plants, the activities of PEPC and NAD-ME of S. lanata under drought stress showed distinct features.

Cytosolic pH as a Secondary Messenger During Light Activation of Phosphoenolpyruvate Carboxylase in Mesophyll Cells of C4 Plants

2001 ◽  
pp. 39-48
Author(s):  
Ch. Bhaskar Rao ◽  
Nasser Syed ◽  
Jhadeswar Murmu ◽  
A. S. Raghavendra
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
C4 Plants ◽  
Light Activation ◽  
Mesophyll Cells ◽  
Cytosolic Ph ◽  
Secondary Messenger

scholarly journals Purification and characterization of cytosolic pyruvate kinase from banana fruit

2000 ◽  
Vol 352 (3) ◽  
pp. 875-882 ◽  
Author(s):  
William L. TURNER ◽  
William C. PLAXTON
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Banana Fruit ◽  
Ph Optimum ◽  
Cytosolic Ph ◽  
Pep Carboxylase ◽  
Sds Page ◽  
Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

Stimulation by autologous serum preheated at 66 °C of the incorporation of [3H]uridine by cultured lymphocytes: comparison with stimulation by concanavalin A

1977 ◽  
Vol 55 (3) ◽  
pp. 215-222 ◽  
Author(s):  
C. M. David ◽  
D. R. Forsdyke
Keyword(s):  
Concanavalin A ◽  
Maximum Velocity ◽  
Autologous Serum ◽  
Rabbit Serum ◽  
Con A ◽  
Rate Limiting Step ◽  
Inhibitory Molecule ◽  
Lymph Node Cells ◽  
Stimulatory Factor ◽  
Rate Limiting

Rabbit serum, preheated at 66 °C for 30 min, produced a stimulation of the incorporation of [3H]uridine by cultured autologous lymph-node cells similar to that caused by concanavalin A (Con-A). However, whereas the percentage stimulation by Con-A increased with time, that by serum preheated at 66 °C (66 °C-serum) reached a peak after 3–4 h and then declined. The decline was greater at higher cell concentrations. Isotope-dilution studies showed that stimulation at 3 h by 66 °C-serum or by Con-A reflected an increase in the maximum velocity of the rate-limiting step for incorporation of [3H]uridine and not a decrease in the pool of uridine and (or) uridine competitors. Experiments in which serum concentration and the relative proportions of serum preheated at 66 °C and serum preheated at 37 °C were varied, suggested that preheating serum at 66 °C removes an inhibitory factor and exposes a stimulatory factor. The stimulatory activity of 66 °C-serum was not dialysable. The results are compatible with a model which requires that lectins activate cultured lymphocytes by influencing the distribution of an inhibitory molecule (perhaps α2-macroglobulin) between the cell surface and the culture medium.

Phosphoenolpyruvate Carboxylase of Thiobacillus thiooxidans. Kinetic and Metabolic Control Properties

1972 ◽  
Vol 50 (2) ◽  
pp. 158-165 ◽  
Author(s):  
R. L. Howden ◽  
H. Lees ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Pep Carboxylase ◽  
Thiobacillus Thiooxidans ◽  
Powerful Activator ◽  
Michaelis Constants ◽  
Noncompetitive Inhibitor ◽  
Decreasing Ph

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) was purified 19-fold from Thiobacillus thiooxidans. The level of enzyme activity was dependent on culture age. No enzyme activity could be obtained from frozen cells.The pH optimum of the enzyme was determined to be around 8.0. Apparent Michaelis constants were determined for the substrates:phosphoenolpyruvate (1.4, 1.5 mM), bicarbonate (0.4, 1.1 mM), and magnesium (1.1, 0.8 mM) at pH 7.0 and 8.0, respectively. Acetyl-CoA was found to be a powerful activator of this enzyme, with the degree of activation increasing with decreasing pH. The concentration of acetyl-CoA to obtain half-maximal activation, however, remained fairly constant and low, namely 1.2 and 1.0 μM at pH 7.0 and 8.0, respectively. L-Aspartate and L-malate were strong inhibitors of enzyme activity. In the presence of aspartate at pH 7.0 the double reciprocal activity plots for PEP became nonlinear, a characteristic of negative cooperativity. These plots became linear with the addition of acetyl-CoA with aspartate now acting as a noncompetitive inhibitor with respect to PEP. At pH 8.0, the same plots were linear with aspartate acting as a competitive inhibitor of PEP. All the other effectors of PEP carboxylase from Salmonella typhimurium and Escherichia coli were found to be ineffective towards the enzyme from T. thiooxidans.

scholarly journals Detecting Photoactivation of Phosphoenolpyruvate Carboxylase in C4 Plants

1985 ◽  
Vol 77 (2) ◽  
pp. 300-302 ◽  
Author(s):  
George Karabourniotis ◽  
Yiannis Manetas ◽  
Nikos A. Gavalas
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
C4 Plants

scholarly journals Glycolate kinase activity in human red cells

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 480-483 ◽  
Author(s):  
S Fujii ◽  
E Beutler
Keyword(s):  
Pyruvate Kinase ◽  
Adenosine Triphosphate ◽  
Kinase Activity ◽  
Maximum Velocity ◽  
Red Cells ◽  
Half Maximum ◽  
Ph Optimum ◽  
Pyruvate Kinase Deficiency ◽  
Low Activity ◽  
Human Red Cells

Abstract Human red cells manifest glycolate kinase activity. This activity copurifies with pyruvate kinase and is decreased in the red cells of subjects with hereditary pyruvate kinase deficiency. Glycolate kinase activity was detected in the presence of FDP or glucose-1,6-P2. In the presence of 1 mmol/L FDP, the Km for adenosine triphosphate (ATP) was 0.28 mmol/L and a half maximum velocity for glycolate was obtained at 40 mmol/L. The pH optimum of the reaction was over 10.5 With 10 mumol/L FDP, 500 mumol/L glucose-1,6-P2, 2 mmol/L ATP, 5 mmol/L MgCl2, and 50 mmol/L glycolate at pH 7.5, glycolate kinase activity was calculated to be approximately 0.0013 U/mL RBC. In view of this low activity even in the presence of massive amounts of glycolate, the glycolate kinase reaction cannot account for the maintenance of the reported phosphoglycolate level in human red cells.

scholarly journals Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties

1978 ◽  
Vol 175 (2) ◽  
pp. 391-406 ◽  
Author(s):  
R Jones ◽  
M B Wilkins ◽  
J R Coggins ◽  
C A Fewson ◽  
A D B Malcolm
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Maximum Velocity ◽  
Single Band ◽  
Kinetic Properties ◽  
Subunit Structure

Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.

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