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

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

Biochemical Journal ◽

10.1042/bj1990603 ◽

1981 ◽

Vol 199 (3) ◽

pp. 603-609 ◽

Cited By ~ 31

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+.

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β-Ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism

Biochemical Journal ◽

10.1042/bj1340239 ◽

1973 ◽

Vol 134 (1) ◽

pp. 239-248 ◽

Cited By ~ 156

Author(s):

Volker Oeding ◽

Hans G. Schlegel

Keyword(s):

Condensation Reaction ◽

Gradient Centrifugation ◽

Substrate Inhibition ◽

Gel Filtration ◽

Deae Cellulose ◽

Saturation Curve ◽

Cleavage Reaction ◽

Acetyl Coa ◽

Freeze Dried ◽

Exclusion Chromatography

1. β-Ketothiolase was purified 49-fold from fructose-grown cells of Hydrogenomonas eutropha H16 with a yield of 27%; the purification procedure involved precipitation by cetyltrimethylammonium bromide, DEAE-cellulose chromatography and exclusion chromatography on Sephadex G-200; the freeze-dried enzyme is stable. The molecular weight determined by sucrose-gradient centrifugation (8.2S) and by gel filtration is 147000–150000. The optimum pH for the cleavage reaction is 8.1, that for the condensation reaction 7.8, both measured in Tris–HCl buffer. 2. The kinetics of the cleavage reaction are described. Substrate-saturation curves were measured with both acetoacetyl-CoA and CoA as the variable substrates. The concentration of the second substrate was kept constant and was varied during successive experiments. The cleavage reaction is characterized by substrate inhibition by acetoacetyl-CoA, which is partially relieved by free CoA. Hill plots indicate two acetoacetyl-CoA-binding sites. 3. The substrate(acetyl-CoA)-saturation curve for the condensation reaction is hyperbolic. The Km was 3.9×10−4m-acetyl-CoA. In the presence of CoA sigmoidal curves were obtained, with an increasing sigmoidicity from 0.03 to 0.30mm-CoA. The inhibitory action of CoA on the β-ketothiolase condensation reaction and its possible involvement in the regulation of poly-β-hydroxybutyrate synthesis and degradation are discussed.

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Structure, mechanism and regulation of pyruvate carboxylase

Biochemical Journal ◽

10.1042/bj20080709 ◽

2008 ◽

Vol 413 (3) ◽

pp. 369-387 ◽

Cited By ~ 230

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.

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Pig liver pyruvate carboxylase. The reaction pathway for the decarboxylation of oxaloacetate

Biochemical Journal ◽

10.1042/bj1390321 ◽

1974 ◽

Vol 139 (2) ◽

pp. 321-329 ◽

Cited By ~ 6

Author(s):

Graham B. Warren ◽

Keith F. Tipton

Keyword(s):

Reaction Mechanism ◽

Pyruvate Carboxylase ◽

Reaction Pathway ◽

Quantitative Description ◽

Preceding Paper ◽

Back Reaction ◽

Acetyl Coa ◽

Pig Liver ◽

Non Linear ◽

Catalytic Cycles

1. The reaction pathway for the decarboxylation of oxaloacetate, 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. MgADP− and Pi bind randomly, at equilibrium, followed by the binding of oxaloacetate. Pyruvate is released before the ordered steay-state release of HCO3− and MgATP2−. 3. These results are entirely consistent with studies on the carboxylation of pyruvate presented in the preceding paper (Warren & Tipton, 1974b) and together they allow a quantitative description of the reaction mechanism of pig liver pyruvate carboxylase. 4. In the absence of other substrates of the back reaction pig liver pyruvate carboxylase will decarboxylate oxaloacetate in a manner that is not inhibited by avidin. 5. Reciprocal plots involving oxaloacetate are non-linear curves, which suggest a negatively co-operative interaction between this substrate and the enzyme.

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

Biochemical Society Transactions ◽

10.1042/bst20120041 ◽

2012 ◽

Vol 40 (3) ◽

pp. 567-572 ◽

Cited By ~ 24

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.

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Probing the ionization state of substrate α-d-glucopyranosyl phosphate bound to glycogen phosphorylase b

Biochemical Journal ◽

10.1042/bj3081017 ◽

1995 ◽

Vol 308 (3) ◽

pp. 1017-1023 ◽

Cited By ~ 1

Author(s):

I P Street ◽

S G Withers

Keyword(s):

Active Site ◽

Glycogen Phosphorylase ◽

Ph Dependence ◽

Substrate Inhibition ◽

19F Nmr ◽

Ionization State ◽

Nmr Studies ◽

Substrate Analogues ◽

Glycogen Phosphorylase B ◽

Pka Value

The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.

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