Characterizing the Molecular Basis of the Allosteric Activation of Pyruvate Carboxylase by Acetyl CoA

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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AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Archaea ◽

10.1155/2006/738517 ◽

2006 ◽

Vol 2 (2) ◽

pp. 95-107 ◽

Cited By ~ 23

Author(s):

Cheryl Ingram-Smith ◽

Kerry S. Smith

Keyword(s):

Phylogenetic Analysis ◽

Molecular Basis ◽

Catalytic Efficiency ◽

Adenosine Monophosphate ◽

Archaeoglobus Fulgidus ◽

Acetyl Coa ◽

Chain Acyl ◽

Key Enzyme ◽

Methanothermobacter Thermautotrophicus ◽

Branched Chain

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS fromMethanothermobacter thermautotrophicus(designated as MT-ACS1) and an ACS fromArchaeoglobus fulgidus(designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.

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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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