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 Structure, mechanism and regulation of pyruvate carboxylase

2008 ◽  
Vol 413 (3) ◽  
pp. 369-387 ◽  
Author(s):  
Sarawut Jitrapakdee ◽  
Martin St Maurice ◽  
Ivan Rayment ◽  
W. Wallace Cleland ◽  
John C. Wallace ◽  
...  
Keyword(s):  
Active Site ◽  
Pyruvate Carboxylase ◽  
Polypeptide Chain ◽  
Tricarboxylic Acid Cycle ◽  
Biotin Carboxylase ◽  
Acetyl Coa ◽  
Specific Expression ◽  
Allosteric Effector ◽  
Bona Fide ◽  
And Function

PC (pyruvate carboxylase) is a biotin-containing enzyme that catalyses the HCO3−- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the biotin carboxylase, carboxyltransferase and biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl-CoA, the biotin moiety transfers the carboxy group between the biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the non-gluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. The present review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme.

scholarly journals Ligand-induced conformational transitions and secondary-structure composition of chicken liver pyruvate carboxylase

1979 ◽  
Vol 177 (2) ◽  
pp. 697-705 ◽  
Author(s):  
Karen S. McGurk ◽  
H. Olin Spivey
Keyword(s):  
Secondary Structure ◽  
Pyruvate Carboxylase ◽  
Conformational Transitions ◽  
Sulphonic Acid ◽  
Chicken Liver ◽  
Stopped Flow ◽  
Acetyl Coa ◽  
Allosteric Effector ◽  
Fluorescence Measurements ◽  
Secondary Structure Composition

Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO3 and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10–13%; ATP, 21–24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO3 had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the ε-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499–3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% β-pleated sheet and 66% other structural types.

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+.

scholarly journals Characterization of the oils present in acid and fermented silages produced from Tilapia filleting residue

2011 ◽  
Vol 40 (2) ◽  
pp. 240-244 ◽  
Author(s):  
Rose Meire Vidotti ◽  
Maria Teresa Bertoldo Pacheco ◽  
Giovani Sampaio Gonçalves
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Nutritional Value ◽  
Lipid Fraction ◽  
Monounsaturated Fatty Acids ◽  
Degree Of Saturation ◽  
Degree Of Hydrolysis ◽  
Animal Feeding ◽  
Hydrolysis Of

The objective of this study was to determine the quality and composition of fatty acid in the lipid fraction of silages obtained from the residue of tilapia processing. Stratification of the lipid layer of the silages occurred at different times among the two types of silage (acid and fermented) and the greatest volume of oil was observed in acid silage (8.67% p/p). Although acid silage was more oxidized, it showed lower contents of free fatty acids probably because the degree of hydrolysis of its components is lower than that of fermented silage. Fatty acid composition did not differ among processes inasmuch as level of ϖ-3 was slightly higher in fermented silage. According to the degree of saturation, monounsaturated fatty acids stood out as the predominant category in acid and fermented silages with values of 39.69% and 33.39%, respectively. The use of antioxidants in the silage is needed because the process of production is carried out at temperatures higher than room temperature. The oil in the silages has excellent nutritional value and contains fatty acids essential for animal feeding.

scholarly journals The existence of multiple tetrameric conformers of chicken liver pyruvate carboxylase and their roles in dilution inactivation

1993 ◽  
Vol 290 (2) ◽  
pp. 583-590 ◽  
Author(s):  
P V Attwood ◽  
W Johannssen ◽  
A Chapman-Smith ◽  
J C Wallace
Keyword(s):  
Rate Constants ◽  
Pyruvate Carboxylase ◽  
Electron Microscopic Observation ◽  
Enzyme Concentration ◽  
Enzymic Activity ◽  
Chicken Liver ◽  
Electron Microscopic ◽  
Acetyl Coa ◽  
Degree Of Dissociation ◽  
Tetrameric Structure

The time-dependent loss of enzymic activity and tetrameric structure of chicken liver pyruvate carboxylase (EC 6.4.1.1) after dilution below 2 units/ml was apparently monophasic and first-order. When examined over a range of initial enzyme concentrations, both activity and tetrameric structure decayed to equilibrium levels which were dependent on the initial concentration. The observed rate constants for the loss of enzymic activity (i) showed no apparent dependence on the initial enzyme concentration, and (ii) were of similar magnitude to the corresponding rate constants of dissociation. Computer simulations of the most likely kinetic model suggest that the predominant form of the dissociated enzyme is the monomer. Dilution of pyruvate carboxylase in the presence of the allosteric activator acetyl-CoA largely prevented the subsequent dissociation of the tetrameric molecule. In addition, acetyl-CoA was able to cause a degree of activation and reassociation when added after dilution inactivation had been allowed to occur. Electron-microscopic observation showed the treatment with avidin before dilution markedly decreased the degree of dissociation of the enzyme tetramer. This structure-stabilizing effect of avidin was dependent on preincubation of the concentrated enzyme solution with acetyl-CoA. We propose that, over a range of protein concentrations, the tetrameric enzyme exists in two forms that are in equilibrium, and that acetyl-CoA alters the equilibrium to favour the more compact form.

scholarly journals Pig liver pyruvate carboxylase. Purification, properties and cation specificity

1974 ◽  
Vol 139 (2) ◽  
pp. 297-310 ◽  
Author(s):  
Graham B. Warren ◽  
Keith F. Tipton
Keyword(s):  
Pyruvate Carboxylase ◽  
Monomer Unit ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Mitochondria ◽  
Univalent Cations ◽  
Acetyl Coa ◽  
Pig Liver ◽  
Apparent Homogeneity ◽  
Polypeptide Chains

1. Pyruvate carboxylase was purified to apparent homogeneity from pig liver mitochondria and shown to be free of all kinetically contaminating enzymes. 2. The enzyme has a mol. wt. of 520000 and is composed of four subunits, each with a mol. wt. of 130000. 3. The enzyme can exist as the active tetramer, dimer and monomer, although the tetramer appears to be the form in which the enzyme is normally assayed. 4. For every 520000g of the enzyme there are 4mol of biotin, 3mol of zinc and 1mol of magnesium. No significant concentrations of manganese were detected. 5. Analysis by sodium dodecyl sulphate–polyacrylamide gel electrophoresis indicates three polypeptide chains per monomer unit, each with a mol. wt. of 47000. 6. The amino acid analysis, stoicheiometry of the reaction and the activity of the enzyme as a function of pH are also presented. 7. The enzyme is activated by a variety of univalent cations but not by Tris+ or triethanolamine+. 8. The activity of the enzyme is dependent on the presence of acetyl-CoA; the low rate in the absence of added acetyl-CoA is not due to an enzyme-bound acyl-CoA. The dissociation constant for enzyme-bound acetyl-CoA is a marked function of pH.

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