scholarly journals Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

2011 ◽  
Vol 509 (2) ◽  
pp. 117-126 ◽  
Author(s):  
Abdussalam Adina-Zada ◽  
Rasmani Hazra ◽  
Chutima Sereeruk ◽  
Sarawut Jitrapakdee ◽  
Tonya N. Zeczycki ◽  
...  
Keyword(s):  
Pyruvate Carboxylase ◽  
Allosteric Activation ◽  
Acetyl Coa ◽  
Allosteric Activator

scholarly journals Factors that influence the translocation of the N-carboxybiotin moiety between the two sub-sites of pyruvate carboxylase

1981 ◽  
Vol 199 (3) ◽  
pp. 603-609 ◽  
Author(s):  
G J Goodall ◽  
G S Baldwin ◽  
J C Wallace ◽  
D B Keech
Keyword(s):  
Dissociation Constant ◽  
Carboxy Group ◽  
Active Site ◽  
Pyruvate Carboxylase ◽  
Alternative Substrate ◽  
Acetyl Coa ◽  
Low Concentrations ◽  
Acid Substrate ◽  
Allosteric Activator ◽  
Rate Of Movement

The active site of pyruvate carboxylase, like those of all biotin-dependent carboxylases, is believed to consist of two spatially distinct sub-sites with biotin acting as a mobile carboxy-group carrier oscillating between the two sub-sites. Some of the factors that influence the location and rate of movement of the N-carboxybiotin were studied. The rate of carboxylation of the alternative substrate, 2-oxobutyrate, was measured at 0 degrees C in an assay system where the isolated enzyme--[14C]carboxybiotin was the carboxy-group donor. The results are consistent with the hypothesis that the location of the carboxybiotin in the active site is determined by the presence of Mg2+, acetyl-CoA and the oxo acid substrate. The presence of Mg2+ favours the holding of the complex at the first sub-site, whereas alpha-oxo acids induce the complex to move to the second sub-site. At low concentrations pyruvate induces this movement but does not efficiently act as a carboxy-group acceptor; hydroxypyruvate, glyoxylate and oxamate, though not carboxylated, still induce the movement. The allosteric activator acetyl-CoA exerts only a slight stimulation on the rate of translocation to the second sub-site, and this stimulation arises from an increase in the dissociation constant for Mg2+.

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

2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Amanda Laseke ◽  
Martin St. Maurice ◽  
Jeremy Lohman ◽  
Aaron Benjamin
Keyword(s):  
Molecular Basis ◽  
Pyruvate Carboxylase ◽  
Allosteric Activation ◽  
Acetyl Coa

Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA

2012 ◽  
Vol 40 (3) ◽  
pp. 567-572 ◽  
Author(s):  
Abdussalam Adina-Zada ◽  
Tonya N. Zeczycki ◽  
Martin St. Maurice ◽  
Sarawut Jitrapakdee ◽  
W. Wallace Cleland ◽  
...  
Keyword(s):  
Active Site ◽  
Pyruvate Carboxylase ◽  
Structural Effects ◽  
Reaction Conditions ◽  
Acetyl Coa ◽  
X Ray ◽  
Allosteric Interactions ◽  
Crystallographic Structures ◽  
And Staphylococcus Aureus ◽  
Allosteric Activator

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.

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.

scholarly journals Pyruvate carboxylase catalysis of phosphate transfer between carbamoyl phosphate and ADP

1991 ◽  
Vol 273 (2) ◽  
pp. 443-448 ◽  
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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