Apparent co-operative effect of acetyl-CoA on pigeon kidney pyruvate carboxylase

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.

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Pig liver pyruvate carboxylase. Purification, properties and cation specificity

Biochemical Journal ◽

10.1042/bj1390297 ◽

1974 ◽

Vol 139 (2) ◽

pp. 297-310 ◽

Cited By ~ 23

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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Effects of biotin on pyruvate carboxylase, acetyl-CoA carboxylase, propionyl-CoA carboxylase, and markers for glucose and lipid homeostasis in type 2 diabetic patients and nondiabetic subjects

American Journal of Clinical Nutrition ◽

10.1093/ajcn/79.2.238 ◽

2004 ◽

Vol 79 (2) ◽

pp. 238-243 ◽

Cited By ~ 29

Author(s):

Armida Báez-Saldaña ◽

Iván Zendejas-Ruiz ◽

Cristina Revilla-Monsalve ◽

Sergio Islas-Andrade ◽

Araceli Cárdenas ◽

...

Keyword(s):

Pyruvate Carboxylase ◽

Lipid Homeostasis ◽

Diabetic Patients ◽

Acetyl Coa Carboxylase ◽

Type 2 Diabetic Patients ◽

Acetyl Coa ◽

Glucose And Lipid Homeostasis ◽

Type 2 Diabetic

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Pyruvate carboxylase catalysis of phosphate transfer between carbamoyl phosphate and ADP

Biochemical Journal ◽

10.1042/bj2730443 ◽

1991 ◽

Vol 273 (2) ◽

pp. 443-448 ◽

Cited By ~ 13

Author(s):

P V Attwood ◽

B D L A Graneri

Keyword(s):

Active Site ◽

Pyruvate Carboxylase ◽

Reverse Reaction ◽

Carbamoyl Phosphate ◽

Acetyl Coa ◽

Essential Requirement ◽

Pyruvate Carboxylation ◽

Ionizable Residues ◽

Ph Profiles

In a reaction that is analogous to the phosphorylation of ADP from carboxyphosphate, pyruvate carboxylase catalyses the formation of ATP from carbamoyl phosphate and ADP at a rate that is about 0.3% of the pyruvate-carboxylation reaction and about 3% of the full reverse reaction. Acetyl-CoA stimulates the phosphorylation of ADP from carbamoyl phosphate but is not an essential requirement of the reaction. Mg2+ also stimulates the reaction, and in the range of Mg2+ concentrations considered the effect of V is much larger in the absence of acetyl-CoA than in its presence. Acetyl-CoA and Mg2+ may be acting in a co-operative way to stimulate the phosphorylation of ADP in a similar way to their effects on the pyruvate-carboxylation reaction. The phosphorylation of ADP by carbamoyl phosphate is also stimulated by the presence of biotin in the part of the active site where this reaction occurs, but again it is not absolutely required for the reaction to proceed. The pH profiles of the phosphorylation of ADP by carbamoyl phosphate indicate that there are at least two ionizable residues involved in the reaction, one of which probably has a role in the release of carbamate from the active site.

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Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase

Biochemistry ◽

10.1021/acs.biochem.7b00383 ◽

2017 ◽

Vol 56 (27) ◽

pp. 3492-3506 ◽

Cited By ~ 4

Author(s):

Lauren E. Westerhold ◽

Lance C. Bridges ◽

Saame Raza Shaikh ◽

Tonya N. Zeczycki

Keyword(s):

Staphylococcus Aureus ◽

Thermodynamic Analysis ◽

Pyruvate Carboxylase ◽

Acetyl Coa ◽

Kinetic And Thermodynamic Analysis

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Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate

Biochemical Journal ◽

10.1042/bj1390311 ◽

1974 ◽

Vol 139 (2) ◽

pp. 311-320 ◽

Cited By ~ 14

Author(s):

Graham B. Warren ◽

Keith F. Tipton

Keyword(s):

Pyruvate Carboxylase ◽

Reaction Pathway ◽

Acetyl Coa ◽

Pig Liver ◽

Equilibrium Exchange ◽

Ping Pong ◽

Magnesium Complexes ◽

The Stability ◽

Catalytic Cycles ◽

Low Rate

1. The reaction pathway for the carboxylation of pyruvate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K+ and acetyl-CoA. 2. Free Mg2+ binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgATP2− binds next, followed by HCO3− and then pyruvate. Oxaloacetate is released before the random release, at equilibrium, of Pi and MgADP−. 3. This reaction pathway is compared with the double displacement (Ping Pong) mechanisms that have previously been described for pyruvate carboxylases from other sources. The reaction pathway proposed for the pig liver enzyme is superior in that it shows no kinetic inconsistencies and satisfactorily explains the low rate of the ATP[unk][32P]Pi equilibrium exchange reaction. 4. Values are presented for the stability constants of the magnesium complexes of ATP, ADP, acetyl-CoA, Pi, pyruvate and oxaloacetate.

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Bicarbonate-dependent ATP cleavage catalysed by pyruvate carboxylase in the absence of pyruvate

Biochemical Journal ◽

10.1042/bj2871011 ◽

1992 ◽

Vol 287 (3) ◽

pp. 1011-1017 ◽

Cited By ~ 20

Author(s):

P V Attwood ◽

B D L A Graneri

Keyword(s):

Reaction Mechanism ◽

Atpase Activity ◽

Active Site ◽

Pyruvate Carboxylase ◽

Substrate Inhibition ◽

Cleavage Reaction ◽

Acetyl Coa ◽

Enzyme Preparations ◽

Pyruvate Carboxylation ◽

Rate Of Movement

Preparations of pyruvate carboxylase catalyse the cleavage of MgATP in the absence of pyruvate and acetyl-CoA. The rate of this cleavage is higher in the presence of HCO3- than in its absence. Incubation of the enzyme preparations with an excess of the pyruvate carboxylase inhibitor, avidin, completely abolishes the pyruvate carboxylating activity of the enzyme preparations but only abolishes the HCO3(-)-dependent MgATP cleaving activity, with no effect on the HCO3(-)-independent ATPase activity. The HCO3(-)-dependent MgATP cleavage is also sensitive to inhibition by a pyruvate carboxylase inhibitor, oxamate, and the dependence of the reaction on the free Mg2+ concentration is similar to that of the pyruvate-carboxylation reaction, whereas the HCO3(-)-independent MgATP cleavage is not dependent on the concentration of free Mg2+ in the range tested. This indicates that MgATP cleavage by pyruvate carboxylase is entirely dependent on the presence of HCO3- and that there may be a low level of ATPase contamination in the enzyme preparations. In addition, inhibition of the HCO3(-)-dependent MgATP cleavage by both avidin and oxamate indicate that although biotin does not directly participate in the reaction, its presence is required in that part of the active site of the enzyme. The rate of HCO3(-)-dependent MgATP cleavage is about 0.07% of that of the full pyruvate carboxylation reaction under similar conditions with saturating substrates. The reaction mechanism is sequential with respect to MgATP and HCO3- addition and Mg2+ adds at equilibrium before MgATP. Acetyl-CoA stimulates the HCO3(-)-dependent MgATP cleavage at low MgATP concentrations, with the stimulation being greater at low Mg2+ concentrations. At high levels of MgATP in the presence of acetyl-CoA, substrate inhibition is evident and is more pronounced at increasing concentrations of Mg2+. This inhibition appears to be, at least in part, caused by inhibition of decarboxylation of the enzyme-carboxybiotin complex by the binding to this complex of Mg2+ and MgATP, which probably act to reduce the rate of movement of carboxybiotin from the site of the MgATP cleavage reaction to that of the pyruvate carboxylation reaction where it is unstable and decarboxylates.

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