Phosphoenolpyruvate Carboxylase of Thiobacillus thioparus. I. General Properties

1975 ◽  
Vol 53 (8) ◽  
pp. 875-880 ◽  
Author(s):  
Daryl J. Hoban ◽  
Ronald M. Lyric
Keyword(s):  
Enzyme Activity ◽  
Phosphoenolpyruvate Carboxylase ◽  
Acetyl Coa ◽  
Pep Carboxylase ◽  
Thiobacillus Thioparus ◽  
General Properties

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxylase (phosphorylating), EC 4.1.1.31) was purified 19-fold from the obligate chemoautotroph, Thiobacillus thioparus.Michaelis constants for the substrates were found to be 0.44 mM for phosphoenolpyruvate, 0.89 mM for bicarbonate, and 0.37 mM for magnesium, using Tris–HCl, pH 7.3.1-Aspartate, 1-malate, and orthophosphate were found to be inhibitors of enzyme activity, while acetyl CoA, FDP, GTP, and CDP had no effect. Dioxane greatly stimulated enzyme activity.

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.

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.

Regulation of Phosphoenolpyruvate Carboxylase in Zea mays by Protein Phosphorylation and Metabolites and Their Roles in Photosynthesis

1996 ◽  
Vol 23 (1) ◽  
pp. 25 ◽  
Author(s):  
Y Gao ◽  
KC Woo
Keyword(s):  
Zea Mays ◽  
Enzyme Activity ◽  
Protein Phosphorylation ◽  
Photosynthetic Rate ◽  
Phosphoenolpyruvate Carboxylase ◽  
Strong Inhibitor ◽  
Physiological Concentration ◽  
Crude Extracts ◽  
Maize Leaves

The effects of metabolites, protein phosphorylation and malate inhibition on phosphoenolpyruvate carboxylase (PEPC) activity were investigated at pH 7.0 in partially purified enzyme from maize leaves. Glycine, glucose 6-phosphate or alanine stimulated the activity two- to three-fold. Glycine and glucose 6-phosphate increased the affinity for PEP by factors of eight and four respectively. These metabolites changed the response of the enzyme activity to pH. Activity increased between pH 6.8 and 8.0 by 10-fold in the absence and 26% in the presence of these metabolites. In vitro phosphorylation of PEPC increased the activity two-fold in the absence but not in the presence of these metabolites. Malate was a strong inhibitor of PEPC, the KI value being 0.25-0.5 mM. Protein phosphorylation and the above metabolites increased the Ki value by factors of three and 12 respectively, but they synergistically increased the Ki 50-fold, thus providing maximal protection against malate inhibition. In the crude extracts from light- and dark-adapted leaves in the presence of a physiological concentration of malate (20 mM), PEPC activity comparable to the photosynthetic rate was obtained only from the light-adapted leaves in the presence of metabolites indicating that both light-induced protein phosphorylation and metabolic activators were essential for PEPC activation during photosynthesis. We propose that both these factors act synergistically to modulate PEPC during photosynthesis in maize.

scholarly journals Acetylation of spermidine and methylglyoxal bis(guanylhydrazone) in baby-hamster kidney cells (BHK-21/C13)

1988 ◽  
Vol 253 (1) ◽  
pp. 223-227 ◽  
Author(s):  
H M Wallace ◽  
M E Nuttall ◽  
F C Robinson
Keyword(s):  
Enzyme Activity ◽  
Proteolytic Degradation ◽  
Nuclear Fraction ◽  
Kidney Cells ◽  
Baby Hamster Kidney ◽  
Acetyl Coa ◽  
Baby Hamster Kidney Cells

Treatment of BHK-21/C13 cells with methylglyoxal bis(guanylhydrazone) (MGBG) induced the cytosolic form of spermidine N1-acetyltransferase. It stabilized the enzyme against proteolytic degradation, but the drug did not affect the enzyme activity in vitro. MGBG was itself acetylated by BHK-21/C13 cells, but at only one-tenth the rate at which spermidine was acetylated. Acetylation occurred almost exclusively in the nuclear fraction. The product was identified as N-acetyl-MGBG by h.p.l.c., by using [3H]acetyl-CoA and [14C]MGBG as co-substrates. The results suggest that the acetylation of MGBG by BHK-21/C13 cells occurs by a different acetyltransferase enzyme from that which acetylates spermidine.

The Sulfite Oxidase of Thiobacillus ferrooxidans (Ferrobacillus ferrooxidans)

1971 ◽  
Vol 49 (10) ◽  
pp. 1125-1130 ◽  
Author(s):  
J. Robie Vestal ◽  
D. G. Lundgren
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Cytochrome C ◽  
Energy Production ◽  
Thiobacillus Ferrooxidans ◽  
Sulfite Oxidase ◽  
Electron Acceptors ◽  
Horse Heart Cytochrome ◽  
Thiobacillus Thioparus ◽  
Horse Heart Cytochrome C

The sulfite oxidase (sulfite: cytochrome c oxidoreductase) from sulfur-grown Thiobacillus ferrooxidans was isolated and partially purified, and its properties were studied. The enzyme was purified 7.3-fold and was 75–85% of the protein present. Sulfite oxidase required SO32− for activity, and could use horse heart cytochrome c and ferricyanide as electron acceptors. The molecular weight was 41 500. The enzyme had a Km for sulfite of 0.58 mM with either ferricyanide or cytochrome c as the electron acceptor. The Km for ferricyanide was 0.25 mM. 5′-AMP did not stimulate enzyme activity. Other properties of the enzyme were similar to the enzyme from Thiobacillus thioparus and Thiobacillus novellus. A metabolic scheme of sulfur utilization for energy production in Thiobacillus ferrooxidans is presented.

Potential Inhibitors of Phosphoenolpyruvate Carboxylase. II. Phosphonic Acid Substrate Analogs Derived From Reaction of Trialkyl Phosphites With Halomethacrylates

1989 ◽  
Vol 42 (2) ◽  
pp. 301 ◽  
Author(s):  
HG Mcfadden ◽  
RLN Harris ◽  
CLD Jenkins
Keyword(s):  
Double Bond ◽  
Nucleophilic Substitution ◽  
Phosphonic Acid ◽  
Phosphoenolpyruvate Carboxylase ◽  
Reaction Conditions ◽  
Pep Carboxylase ◽  
Substrate Analogs ◽  
Trialkyl Phosphites ◽  
Potential Inhibitors ◽  
Acid Substrate

Analogues of phosphoenolpyruvate (PEP), the substrate of PEP carboxylase , were synthesized as potential inhibitors of the enzyme. Esterified analogue precursors were obtained by nucleophilic substitution of various halomethacrylate derivatives with trialkyl phosphites. Substitution of the halomethacrylates can occur either α to the leaving halogen or in the γ position with subsequent allylic shift of the double bond. The course of the reaction was influenced both by reaction conditions and the nature of the substituents on the reactants. The phosphonomethacrylates obtained were hydrolysed to PEP analogues that were found to be moderate to good inhibitors of PEP carboxylase. Phosphonomethacrylic acid derivatives bearing two halogen substituents in the γ position were found to be the most potent inhibitors of this enzyme.

Phosphoenolpyruvate carboxylase activity from Avena coleoptile tissue. Regulation by H+ and malate

1978 ◽  
Vol 56 (4) ◽  
pp. 404-407 ◽  
Author(s):  
B. C. Hill ◽  
A. W. Bown
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Enzyme System ◽  
Mass Action ◽  
Direct Inhibition ◽  
Carboxylase Activity ◽  
Pep Carboxylase ◽  
Action Effect ◽  
Avena Coleoptile ◽  
Oxaloacetic Acid ◽  
Regulatory Properties

Preparations of phosphoenolpyruvate (PEP) carboxylase activity from Avenu sativa coleoptile tissue were assayed by measuring the incorporation of labelled bicarbonate into a derivative of oxaloacetic acid or by coupling oxaloacetic acid production to malate dehydrogenase activity and the oxidation of NADH. Malate inhibition of PEP carboxylase activity was found to be noncompetitive, was not due to a mass action effect on the coupled enzyme system or to chelation of Mg2+, and probably involved direct inhibition of the enzyme by malate. Maximal PEP carboxylase activity was exhibited around pH 8.0 and increased 125% between pH 7.0 and pH 7.6. Inhibition by 4 mML-malate was virtually complete at pH 7.0 and decreased to 10% inhibition at pH 8. This information is discussed in the light of data which demonstrates that in response to IAA. coleoptile tissue accumulates malate and secretes H+. The regulatory properties of PEP carboxylase are consistent with a role in malate production which could resist increases in intracellular pH resulting from an auxin-stimulated H+ efflux.

scholarly journals Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

1999 ◽  
Vol 181 (4) ◽  
pp. 1088-1098 ◽  
Author(s):  
Castor Menendez ◽  
Zsuzsa Bauer ◽  
Harald Huber ◽  
Nasser Gad’on ◽  
Karl-Otto Stetter ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Carbon Fixation ◽  
Coenzyme A ◽  
Autotrophic Growth ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

ABSTRACT The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed forC. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus andAcidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula,S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.

scholarly journals Factors which affect the activity of purified rat liver acyl-CoA oxidase

1993 ◽  
Vol 290 (1) ◽  
pp. 97-102 ◽  
Author(s):  
R Hovik ◽  
H Osmundsen
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Substrate Inhibition ◽  
Critical Micellar Concentration ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Acyl Coa Oxidase ◽  
Triton X

The activity of the enzyme acyl-CoA oxidase (EC 1.3.99.3) is influenced by detergents. At concentrations above the critical micellar concentration, Triton X-100, Triton X-114 and Thesit stimulate oxidase activity. Lower concentrations of Triton X-100 and Triton X-114 render the acyl-CoA oxidase less sensitive towards substrate inhibition by palmitoyl-CoA or dec-4-cis-enoyl-CoA. Other detergents inhibited the enzyme activity. CoA was found to be a relatively powerful competitive inhibitor of the enzyme, with a Ki, slope value of 63 +/- 3 microM. This inhibition is dependent on an intact CoA molecule, as dephospho-CoA, dethio-CoA and acetyl-CoA are less potent inhibitors of the enzyme. Dec-2-trans-enoyl-CoA is a product-inhibitor of acyl-CoA oxidase, with a Ki, slope value of 7 +/- 1 microM.

scholarly journals Hormonal regulation of adipose-tissue acetyl-coenzyme A carboxylase by changes in the polymeric state of the enzyme. The role of long-chain fatty acyl-coenzyme A thioesters and citrate

1974 ◽  
Vol 142 (2) ◽  
pp. 365-377 ◽  
Author(s):  
Andrew P. Halestrap ◽  
Richard M. Denton
Keyword(s):  
Enzyme Activity ◽  
Coenzyme A ◽  
Total Activity ◽  
Fatty Acyl ◽  
Acetyl Coa Carboxylase ◽  
Initial Activity ◽  
Acetyl Coa ◽  
Coa Thioesters ◽  
Fat Pads

1. Acetyl-CoA carboxylase activity was measured in extracts of rat epididymal fat-pads either on preparation of the extracts (initial activity) or after incubation of the extracts with citrate (total activity). In the presence of glucose or fructose, brief exposure of pads to insulin increased the initial activity of acetyl-CoA carboxylase; no increase occurred in the absence of substrate. Adrenaline in the presence of glucose and insulin decreased the initial activity. None of these treatments led to a substantial change in the total activity of acetyl-CoA carboxylase. A large decrease in the initial activity of acetyl-CoA carboxylase also occurred with fat-pads obtained from rats that had been starved for 36h although the total activity was little changed by this treatment. 2. Conditions of high-speed centrifugation were found which appear to permit the separation of the polymeric and protomeric forms of the enzyme in fat-pad extracts. After the exposure of the fat-pads to insulin (in the presence of glucose), the proportion of the enzyme in the polymeric form was increased, whereas exposure to adrenaline (in the presence of glucose and insulin) led to a decrease in enzyme activity. 3. These changes are consistent with a role of citrate (as activator) or fatty acyl-CoA thioesters (as inhibitors) in the regulation of the enzyme by insulin and adrenaline; no evidence that the effects of these hormones involve phosphorylation or dephosphorylation of the enzyme could be found. 4. Changes in the whole tissue concentration of citrate and fatty acyl-CoA thioesters were compared with changes in the initial activity of acetyl-CoA carboxylase under a variety of conditions of incubation. No correlation between the citrate concentration and the initial enzyme activity was evident under any condition studied. Except in fat-pads which were exposed to insulin there was little inverse correlation between the concentration in the tissue of fatty acyl-CoA thioesters and the initial activity of acetyl-CoA carboxylase. 5. It is suggested that changes in the concentration of free fatty acyl-CoA thioesters (which may not be reflected in whole tissue concentrations of these metabolites) may be important in the regulation of the activity of acetyl-CoA carboxylase. The possibility is discussed that the concentration of free fatty acyl-CoA thioesters may be controlled by binding to a specific protein with properties similar to albumin.

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