Jorge Babul - Academia.edu (original) (raw)

Papers by Jorge Babul

Research paper thumbnail of Effect of ATP on phosphofructokinase-2 from Escherichia coli. A mutant enzyme altered in the allosteric site for MgATP

Journal of Biological Chemistry, Sep 1, 1985

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Research paper thumbnail of A phosphofructokinase mutant of Escherichia coli altered in its allosteric properties impairs gluconeogenic growth

PubMed, Dec 1, 1985

The regulation of metabolic fluxes is accomplished by modulation of key enzyme catalyzed reaction... more The regulation of metabolic fluxes is accomplished by modulation of key enzyme catalyzed reactions. This modulation takes place partially through the control of the catalytic activity of enzymes labelled as regulatory enzymes. The kinetic behavior of many regulatory enzymes can be explained in terms of multiple binding sites for effector molecules. Of these, the ones that play their control over catalysis by binding at an allosteric site have been considered of much importance. Nevertheless, proof that the effects observed in vitro, are in fact responsible for the physiological regulation in vivo, is scarce. In this regard, mutant enzymes altered in their allosteric properties might be useful. This will be illustrated with an enzyme considered crucial for the regulation of carbohydrate metabolism, namely phosphofructokinase. We present here the comparison of some of the kinetic and structural properties of wild type phosphofructokinase-2 of E. coli and of a mutant form which impairs gluconeogenic growth, an indication of the significance of the in vivo regulation. The main differences between the enzymes are their kinetic reaction mechanism, inhibitability by ATP, and aggregation states in the presence of substrates and effectors. So far these differences support only speculations as to the mechanism of the gluconeogenic impairment observed in strains that contain the mutant enzyme, a few of which are offered.

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Research paper thumbnail of Crystal structure of the phosphofructokinase-2 from Escherichia coli in complex with fructose-6-phosphate

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Research paper thumbnail of Ensemble Switching of the DNA-Binding Domain of Human FoxP1

Biophysical Journal, 2021

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Research paper thumbnail of Structural Dynamics of the Heterodimerization Between the DNA-Binding Domains from Human FoxP1 and FoP2 Transcription Factors

Biophysical Journal, 2021

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Research paper thumbnail of New visible and selective DNA staining method in gels with tetrazolium salts

Analytical Biochemistry, 2017

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Research paper thumbnail of Crystal structure of the Phosphofructokinase-2 mutant Y23D from Escherichia coli

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Research paper thumbnail of The alkaline isomerization of lysine-modified ferricytochrome c

Biochimica et Biophysica Acta (BBA) - Protein Structure, 1975

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Research paper thumbnail of An Alteration in Phosphofructokinase 2 of Escherichia coli which Impairs Gluconeogenic Growth and Improves Growth on Sugars

European Journal of Biochemistry, 2005

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is ... more Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.

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Research paper thumbnail of An in vitro model showing different rates of substrate cycle for phosphofructokinases of Escherichia coli with different kinetic properties

European Journal of Biochemistry, 1991

An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fruc... more An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fructose-bisphosphatase. The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2), and a mutant form of phosphofructokinase-2 (PFK-2*). Results show that for a variety of conditions the PFK-1/fructose-bisphosphatase pair gives the lowest and the PFK-2*/fructose-bisphosphatase pair the highest rates of substrate cycle, with the PFK-2/fructose-bisphosphatase pair in an intermediate position. The effects of variables such as maximum activity ratios and MgATP concentration were explored. The possible role of MgATP in decreasing the futile cycle of the PFK-2/fructose-bisphosphatase pair is described. The results are discussed in terms of possible metabolic consequences of PFK-2* and of predictions of the model to be tested in vivo.

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Research paper thumbnail of Are the Aerobic and Anaerobic Phosphofructokinases of Escherichia coli Different?

European Journal of Biochemistry, 1977

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Research paper thumbnail of An examination of the involvement of proline peptide isomerization in protein folding

Journal of Molecular Biology, 1978

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Research paper thumbnail of Reversible unfolding of dimeric phosphofructokinase-2 from Escherichia coli reveals a dominant role of inter-subunit contacts for stability

FEBS Letters, 2009

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Research paper thumbnail of The existence of heme-protein coordinate-covalent bonds in denaturing solvents

Biopolymers, 1971

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Research paper thumbnail of Unfolding Pathway of the Dimeric and Tetrameric Forms of Phosphofructokinase-2 from Escherichia coli †

Biochemistry, 2007

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Research paper thumbnail of Phosphate modification of fructose-1,6-bisphosphate aldolase in Escherichia coli

Biochemical and Biophysical Research Communications, 1988

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Research paper thumbnail of The structure of hemopeptide 1–65 from cytochrome c

Archives of Biochemistry and Biophysics, 1972

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Research paper thumbnail of Influence of ligands on the aggregation of the normal and mutant forms of phosphofructokinase 2 of Escherichia coli

Archives of Biochemistry and Biophysics, 1988

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Research paper thumbnail of Fructose bisphosphatase from Escherichia coli. Purification and characterization

Archives of Biochemistry and Biophysics, 1983

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Research paper thumbnail of Regulatory network of the allosteric ATP inhibition of E. coli phosphofructokinase-2 studied by hybrid dimers

Biochimie, 2016

We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of dif... more We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of different forms of the enzyme together with a kinetic enzyme analysis. Here we complement the mechanism by using hybrid oligomers of the homodimeric enzyme to get insights about the allosteric communication pathways between the same sites or different ones located in different subunits. Kinetic analysis of the hybrid enzymes indicate that homotropic interactions between allosteric sites for ATP or between substrate sites for fructose-6-P have a minor effect on the enzymatic inhibition induced by ATP. In fact, the sigmoid response for fructose-6-P observed at elevated ATP concentrations can be eliminated even though the enzymatic inhibition is still operative. Nevertheless, leverage coupling analysis supports heterotropic interactions between the allosteric ATP and fructose-6-P binding occurring between and within each subunit.

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Research paper thumbnail of Effect of ATP on phosphofructokinase-2 from Escherichia coli. A mutant enzyme altered in the allosteric site for MgATP

Journal of Biological Chemistry, Sep 1, 1985

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Research paper thumbnail of A phosphofructokinase mutant of Escherichia coli altered in its allosteric properties impairs gluconeogenic growth

PubMed, Dec 1, 1985

The regulation of metabolic fluxes is accomplished by modulation of key enzyme catalyzed reaction... more The regulation of metabolic fluxes is accomplished by modulation of key enzyme catalyzed reactions. This modulation takes place partially through the control of the catalytic activity of enzymes labelled as regulatory enzymes. The kinetic behavior of many regulatory enzymes can be explained in terms of multiple binding sites for effector molecules. Of these, the ones that play their control over catalysis by binding at an allosteric site have been considered of much importance. Nevertheless, proof that the effects observed in vitro, are in fact responsible for the physiological regulation in vivo, is scarce. In this regard, mutant enzymes altered in their allosteric properties might be useful. This will be illustrated with an enzyme considered crucial for the regulation of carbohydrate metabolism, namely phosphofructokinase. We present here the comparison of some of the kinetic and structural properties of wild type phosphofructokinase-2 of E. coli and of a mutant form which impairs gluconeogenic growth, an indication of the significance of the in vivo regulation. The main differences between the enzymes are their kinetic reaction mechanism, inhibitability by ATP, and aggregation states in the presence of substrates and effectors. So far these differences support only speculations as to the mechanism of the gluconeogenic impairment observed in strains that contain the mutant enzyme, a few of which are offered.

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Research paper thumbnail of Crystal structure of the phosphofructokinase-2 from Escherichia coli in complex with fructose-6-phosphate

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Ensemble Switching of the DNA-Binding Domain of Human FoxP1

Biophysical Journal, 2021

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Research paper thumbnail of Structural Dynamics of the Heterodimerization Between the DNA-Binding Domains from Human FoxP1 and FoP2 Transcription Factors

Biophysical Journal, 2021

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Research paper thumbnail of New visible and selective DNA staining method in gels with tetrazolium salts

Analytical Biochemistry, 2017

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Research paper thumbnail of Crystal structure of the Phosphofructokinase-2 mutant Y23D from Escherichia coli

Bookmarks Related papers MentionsView impact

Research paper thumbnail of The alkaline isomerization of lysine-modified ferricytochrome c

Biochimica et Biophysica Acta (BBA) - Protein Structure, 1975

Bookmarks Related papers MentionsView impact

Research paper thumbnail of An Alteration in Phosphofructokinase 2 of Escherichia coli which Impairs Gluconeogenic Growth and Improves Growth on Sugars

European Journal of Biochemistry, 2005

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is ... more Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.

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Research paper thumbnail of An in vitro model showing different rates of substrate cycle for phosphofructokinases of Escherichia coli with different kinetic properties

European Journal of Biochemistry, 1991

An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fruc... more An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fructose-bisphosphatase. The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2), and a mutant form of phosphofructokinase-2 (PFK-2*). Results show that for a variety of conditions the PFK-1/fructose-bisphosphatase pair gives the lowest and the PFK-2*/fructose-bisphosphatase pair the highest rates of substrate cycle, with the PFK-2/fructose-bisphosphatase pair in an intermediate position. The effects of variables such as maximum activity ratios and MgATP concentration were explored. The possible role of MgATP in decreasing the futile cycle of the PFK-2/fructose-bisphosphatase pair is described. The results are discussed in terms of possible metabolic consequences of PFK-2* and of predictions of the model to be tested in vivo.

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Are the Aerobic and Anaerobic Phosphofructokinases of Escherichia coli Different?

European Journal of Biochemistry, 1977

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Research paper thumbnail of An examination of the involvement of proline peptide isomerization in protein folding

Journal of Molecular Biology, 1978

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Reversible unfolding of dimeric phosphofructokinase-2 from Escherichia coli reveals a dominant role of inter-subunit contacts for stability

FEBS Letters, 2009

Bookmarks Related papers MentionsView impact

Research paper thumbnail of The existence of heme-protein coordinate-covalent bonds in denaturing solvents

Biopolymers, 1971

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Unfolding Pathway of the Dimeric and Tetrameric Forms of Phosphofructokinase-2 from Escherichia coli †

Biochemistry, 2007

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Phosphate modification of fructose-1,6-bisphosphate aldolase in Escherichia coli

Biochemical and Biophysical Research Communications, 1988

Bookmarks Related papers MentionsView impact

Research paper thumbnail of The structure of hemopeptide 1–65 from cytochrome c

Archives of Biochemistry and Biophysics, 1972

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Influence of ligands on the aggregation of the normal and mutant forms of phosphofructokinase 2 of Escherichia coli

Archives of Biochemistry and Biophysics, 1988

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Fructose bisphosphatase from Escherichia coli. Purification and characterization

Archives of Biochemistry and Biophysics, 1983

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Regulatory network of the allosteric ATP inhibition of E. coli phosphofructokinase-2 studied by hybrid dimers

Biochimie, 2016

We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of dif... more We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of different forms of the enzyme together with a kinetic enzyme analysis. Here we complement the mechanism by using hybrid oligomers of the homodimeric enzyme to get insights about the allosteric communication pathways between the same sites or different ones located in different subunits. Kinetic analysis of the hybrid enzymes indicate that homotropic interactions between allosteric sites for ATP or between substrate sites for fructose-6-P have a minor effect on the enzymatic inhibition induced by ATP. In fact, the sigmoid response for fructose-6-P observed at elevated ATP concentrations can be eliminated even though the enzymatic inhibition is still operative. Nevertheless, leverage coupling analysis supports heterotropic interactions between the allosteric ATP and fructose-6-P binding occurring between and within each subunit.

Bookmarks Related papers MentionsView impact