Pyruvate carboxylase from Thiobacillus novellus: properties and possible function

1984 ◽  
Vol 30 (5) ◽  
pp. 532-539 ◽  
Author(s):  
A. M. Charles ◽  
D. W. Willer
Keyword(s):  
Initial Velocity ◽  
Pyruvate Carboxylase ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Citrate Cycle ◽  
End Products ◽  
Sigmoidal Curve ◽  
Hill Coefficients

Pyruvate carboxylase (EC 6.4.1.1) from Thiobacillus novellus (ATCC 8093) was highly purified and found to have a pH optimum of 7.6, a temperature optimum of 25–35 °C, and a requirement for a monovalent and a divalent cation, as well as a CoA derivative, for maximum activity. These were best served by K+, Mg2+, and acetyl-CoA. Km values for pyruvate, ATP, HCO3− and Mg2+ were 0.25, 0.04, 0.27, and 0.44 mM, respectively. Initial velocity plots of increasing acetyl-CoA concentrations gave a sigmoidal curve with Ka of 4.2 μM, and Hill coefficients of 2.2. Plots of fixed acetyl-CoA concentrations against varying concentrations of pyruvate, ATP, or CO2 all gave rectangular hyperbolae. Apart from end products, only hydroxypyruvic acid was found to be inhibitory. The enzyme was very sensitive to mercurials. This enzyme is not believed to serve an anaplerotic function, because of the simultaneous presence of the highly regulated phosphoenolpyruvate carboxylase in the organism. Instead, it may function either to supply oxaloacetate to the citrate cycle or as part of the system that provides reduced NADP+.

AN ISOPHENOXAZINE SYNTHASE FROM PYGNOPORUS COCCINEUS (FR.) BOND. AND SING.

1964 ◽  
Vol 42 (11) ◽  
pp. 1515-1526 ◽  
Author(s):  
P. M. Nair ◽  
L. C. Vining
Keyword(s):  
Electron Acceptor ◽  
Specific Activity ◽  
Fold Increase ◽  
Maximum Activity ◽  
Flavin Mononucleotide ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Hydroxyanthranilic Acid ◽  
Pycnoporus Coccineus ◽  
Citrate Phosphate

Mycelium from cultures of the red polypore Pycnoporus coccineus (Fr.) Bond. and Sing, contains an enzyme which catalyzes the oxidative condensation of 2 molecules of 2-aminophenol to yield 2-amino-3H-isophenoxazin-3-one. Fractionation of the crude extract has given a preparation with an 893-fold increase in specific activity. The purified enzyme has a pH optimum in citrate-phosphate buffer of 5.0, and a temperature optimum of 55°. The Km value is 4.35 × 10−4 M. FMN and Mn++ ions were required for maximum activity. FAD also served as an electron acceptor. Of the metal ions tested only Mn++ activated the reaction. Hg++ and Fe++ inhibited strongly. The course of the reaction when cofactors were added separately suggested that flavin mononucleotide is the initial electron acceptor and that Mn++ ions are required for reoxidation of the flavin. The enzyme has narrow specificity, and does not catalyze the oxidation of 3-hydroxyanthranilic acid, 3-hydroxykynurenine, or pyrocatechol.

scholarly journals Purification and properties of a 1,3-1,4-β-d-glucanase (lichenase, 1,3-1,4-β-d-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli

1988 ◽  
Vol 255 (3) ◽  
pp. 833-841 ◽  
Author(s):  
J D Erfle ◽  
R M Teather ◽  
P J Wood ◽  
J E Irvin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Glucanase Activity ◽  
Ph Optimum ◽  
Beta Glucanase ◽  
Purification And Properties ◽  
Hydrolysis Of

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.

scholarly journals The Pyruvate Carboxylase of Verticillium albo-atrum

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 15-19 ◽  
Author(s):  
R. E. Hartman ◽  
N. T. Keen
Keyword(s):  
Pyruvate Carboxylase ◽  
Krebs Cycle ◽  
Physiological Role ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Carboxylase Activity ◽  
Metabolic Intermediates ◽  
Krebs Cycle Intermediates ◽  
Verticillium Albo Atrum

The pyruvate carboxylase of Verticillium albo-atrum had a pH optimum of 7·8 and a specific requirement for ATP. At the optimum pH, magnesium ions were required for maximum activity, while at pH 6·8 manganese was more effective than magnesium. Potassium was stimulatory while sodium was ineffective. Avidin and p-chloromercuribenzoate strongly inhibited the enzyme while biotin and dithiothreitol, respectively, reversed the effect of the inhibitors. Aspartate and oxalacetate were inhibitory while acetyl-CoA and CoA reversed the inhibition by aspartate. These cofactors were ineffective in the absence of aspartate. None of the tested metabolic intermediates was stimulatory to pyruvate carboxylase activity while NADP+ and 2,3-diphosphoglycerate were the most effective inhibitors (75%) at a concentration of 6·7 mM. Levels of pyruvate carboxylase in cells grown on glucose, acetate, malate, xylose, glycerol or aspartate differed only slightly. The data indicated that the physiological role of pyruvate carboxylase in V. albo-atrum is the anaplerotic biosynthesis of C4 Krebs-cycle intermediates from pyruvate.

scholarly journals Purification and Characterization of Two Extremely Thermostable Enzymes, Phosphate Acetyltransferase and Acetate Kinase, from the Hyperthermophilic Eubacterium Thermotoga maritima

1999 ◽  
Vol 181 (6) ◽  
pp. 1861-1867 ◽  
Author(s):  
Anne-Katrin Bock ◽  
Jürgen Glasemacher ◽  
Roland Schmidt ◽  
Peter Schönheit
Keyword(s):  
Molecular Mass ◽  
Thermotoga Maritima ◽  
Enzyme Reaction ◽  
Acetyl Phosphate ◽  
Temperature Optimum ◽  
Terminal Amino Acid ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Terminal Amino ◽  
Phosphate Acetyltransferase

ABSTRACT Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (α4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases fromClostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi ⇌ acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 μM, respectively; the apparentV max values (at 55°C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90°C and was not inactivated by heat upon incubation at 80°C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (α2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP ⇌ acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent V max values (at 50°C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90°C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4(1 M), which was most effective, the enzyme did not lose activity upon incubation at 100°C for 3 h. The temperature optimum at 90°C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

scholarly journals The atypical velocity response by pyruvate carboxylase to increasing concentrations of acetyl-coenzyme A

1979 ◽  
Vol 179 (3) ◽  
pp. 497-502 ◽  
Author(s):  
Simon B. Easterbrook-Smith ◽  
Anne J. Campbell ◽  
D. Bruce Keech ◽  
John C. Wallace
Keyword(s):  
Velocity Profile ◽  
Pyruvate Carboxylase ◽  
Degree Of Saturation ◽  
Acetyl Coa ◽  
Reaction Velocity ◽  
Low Protein ◽  
Allosteric Effector ◽  
Velocity Response ◽  
Hill Coefficients ◽  
Hydrolysis Of

An investigation was made of the interaction of pyruvate carboxylase with its allosteric effector, acetyl-CoA, and the velocity profile of the deacylation of acetyl-CoA as a function of acetyl-CoA concentration indicated that this ligand does not bind to this enzyme in a positive homotropic co-operative manner. An examination was therefore made of the factors that contribute to the sigmoidicity of the rate curves obtained for pyruvate carboxylation with various concentrations of acetyl-CoA. Hill coefficients for acetyl-CoA obtained with both sheep and chicken liver pyruvate carboxylases were found to be dependent on the fixed pyruvate concentration used in the assay solution. Thus, by varying the acetyl-CoA concentration, the degree of saturation of the enzyme by pyruvate was also changed. A further consequence of non-saturating concentrations of pyruvate was that the non-productive hydrolysis of the enzyme– carboxybiotin complex increased, resulting in an under-estimate of the reaction velocity measured by oxaloacetate formation. Another factor contributing to the sigmoidicity is that, at non-saturating concentrations of acetyl-CoA, the enzyme undergoes inactivation upon dilution to low protein concentrations, again resulting in an under-estimate of the reaction velocity. Under conditions where none of the above factors was operating and the only effect of varying acetyl-CoA concentrations was to alter the proportion of the enzyme catalysing the carboxylation reaction at acetyl-CoA-dependent and -independent rates, the sigmoidicity of the acetyl-CoA velocity profile was completely eliminated.

scholarly journals Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Mechanism and control of the reaction

1971 ◽  
Vol 122 (5) ◽  
pp. 663-669 ◽  
Author(s):  
T. K. Sundaram ◽  
J. J. Cazzulo ◽  
H. L. Kornberg
Keyword(s):  
Pyruvate Carboxylase ◽  
Thermal Denaturation ◽  
Bacillus Stearothermophilus ◽  
The Other ◽  
Acetyl Coa ◽  
Allosteric Effector ◽  
Allosteric Effectors ◽  
Hill Coefficients ◽  
And Control ◽  
The Hill

1. Acetyl-CoA acts as a positive allosteric effector in the formation of active pyruvate carboxylase from its apoenzyme, ATP and (+)-biotin which is catalysed by holoenzyme synthetase; this effect is counteracted by l-aspartate. 2. The Hill coefficients (apparent n values) were approximately 2 for acetyl-CoA and 4 for l-aspartate; the n value for each effector remained constant when the concentration of the other effector was varied. 3. Active pyruvate carboxylase was formed also when the apoenzyme was incubated with holoenzyme synthetase and synthetic biotinyl-5′-AMP; acetyl-CoA and l-aspartate affected this process as they did the overall reaction from (+)-biotin and ATP. 4. When hydroxylamine replaced the apoenzyme, holoenzyme synthetase catalysed the formation of biotinylhydroxamate from (+)-biotin and ATP. This reaction was not affected by the allosteric effectors. 5. The apoenzyme was protected against thermal denaturation by acetyl-CoA and, to a lesser degree, by l-aspartate. The holoenzyme synthetase was not markedly protected by these effectors. 6. It is concluded that the allosteric effectors act on the apoenzyme and not the synthetase.

scholarly journals Metabolism of pyruvate and malate by isolated fat-cell mitochondria

1971 ◽  
Vol 125 (1) ◽  
pp. 105-113 ◽  
Author(s):  
B. R. Martin ◽  
R. M. Denton
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Carboxylase ◽  
Adenine Nucleotides ◽  
Acid Synthesis ◽  
Fat Cells ◽  
Fat Cell ◽  
Pyruvate Metabolism ◽  
Flow Rates ◽  
Citrate Cycle ◽  
End Products

1. Metabolism of pyruvate and malate by isolated fat-cell mitochondria incubated in the presence of ADP and phosphate has been studied by measuring rates of pyruvate uptake, malate utilization or production, citrate production and oxygen consumption. From these measurements calculations of the flow rates through pyruvate carboxylase, pyruvate dehydrogenase and citrate cycle have been made under various conditions. 2. In the presence of bicarbonate, pyruvate was largely converted into citrate and malate and only about 10% was oxidized by the citrate cycle; citrate and malate outputs were linear after lag periods of 6–9min and 3min respectively, and no other end products of pyruvate metabolism were detected. On the further addition of malate or hydroxymalonate, the lag in the rate of citrate output was less marked but no net malate disappearance was detected. If, however, bicarbonate was omitted then net malate uptake was observed. Addition of butyl malonate was found to greatly inhibit the metabolism of pyruvate to citrate and malate in the presence of bicarbonate. 3. These results are in agreement with earlier conclusions that in adipose tissue acetyl units for fatty acid synthesis are transferred to the cytoplasm as citrate and that this transfer requires malate presumably for counter transport. They also support the view that oxaloacetate for citrate synthesis is preferentially formed from pyruvate through pyruvate carboxylase rather than malate through malate dehydrogenase and that the mitochondrial metabolism of citrate in fat-cells is restricted. The possible consequences of these conclusions are discussed. 4. Studies on the effects of additions of adenine nucleotides to pyruvate metabolism by isolated fat-cell mitochondria are consistent with inhibition of pyruvate carboxylase in the presence of ADP and pyruvate dehydrogenase in the presence of ATP.

Biochemical studies on the production of neuraminidase by environmental isolates of Vibrio cholerae non-O1 from Bulgaria

2011 ◽  
Vol 57 (7) ◽  
pp. 606-610 ◽  
Author(s):  
Rumyana Eneva ◽  
Stephan Engibarov ◽  
Tanya Strateva ◽  
Radoslav Abrashev ◽  
Ignat Abrashev
Keyword(s):  
Vibrio Cholerae ◽  
Pathogenic Bacteria ◽  
Environmental Isolates ◽  
Anaerobic Conditions ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Cholera Vibrio ◽  
Key Factor ◽  
Aeration Conditions ◽  
The One

Neuraminidase is a key factor in the infectious process of many viruses and pathogenic bacteria. The neuraminidase enzyme secreted by the etiological agent of cholera — Vibrio cholerae О1 — is well studied in contrast with the one produced by non-O1/non-O139 V. cholerae. Environmental non-O1/non-O139 V. cholerae isolates from Bulgaria were screened for production of neuraminidase. The presence of the neuraminidase gene nanH was detected in 18.5% of the strains. Тhe strain showing highest activity (30 U/mL), V. cholerae non-O1/13, was used to investigate the enzyme production in several media and at different aeration conditions. The highest production of extracellular neuraminidase was observed under microaerophilic conditions, which is possibly related to its role in the infection of intestine epithelium, where the oxygen content is low. On the other hand, this is another advantage of the microbe in such microaerophilic environments as sediments and lake mud. The highest production of intracellular neuraminidase was observed at anaerobic conditions. The ratio of extracellular to intracellular neuraminidase production in V. cholerae was investigated. The temperature optimum of the enzyme was determined to be 50 °C and the pH optimum to be 5.6–5.8.

Pyridine–Adenine Dinucleotide Transhydrogenase Activity in Cells Cultured from Rat Hepatoma

1972 ◽  
Vol 50 (5) ◽  
pp. 447-456 ◽  
Author(s):  
C. De Luca ◽  
R. P. Gioeli
Keyword(s):  
Phosphate Buffer ◽  
Pyridine Nucleotide ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Apparent Lack ◽  
Minimal Deviation ◽  
Ph Range ◽  
Pyridine Nucleotide Transhydrogenase ◽  
Rat Hepatoma ◽  
Cells Cultured

Preparations from cells cultured from a minimal-deviation hepatoma in the rat exhibit pyridine nucleotide transhydrogenase (NAD(P)H: NAD(P) oxidoreductase, EC 1.6.1.1) activity. The pH optimum, its release by digitonin, and its apparent lack of dependence on steroids for activity tentatively classify it as a transhydrogenase of the type first described for animal tissue.Enzyme preparations from digitonin-treated homogenates were very unstable. The time necessary for the loss of one-half the activity was 16–18 h when the enzyme was stored at 5 °C; this was reduced to 4 h when storage was in polycarbonate tubes.The enzyme apparently transferred hydrogen directly and with equal ease from NADH to both the 3-acetyl-pyridine and thionicotinamide analogues of NAD. Half-saturation values for NAD and its acetylpyridine analogue were 0.99 × 10−5 M and 3.55 × 10−4 M, respectively. The enzyme exhibited its maximum activity in phosphate buffer at pH 5.8. It was inhibited by 50–60% over the pH range 7.0–8.5 in Tris buffer. This could be reversed by dithiothreitol; reversal was complete between pH 8.0 and 8.5.

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