Functional complementation of yeast phosphofructokinase mutants by the non‐allosteric enzyme from <i>Dictyostelium discoideum</i>

Estévez, Antonio M.; Heinisch, Jürgen J.; Aragón, J. L.

doi:10.1016/0014-5793(95)01085-s

Cited by 22 publications

(21 citation statements)

References 32 publications

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“…The results obtained here are supported by experiments on the heterologous expression of the Pfk from Dictyostelium discoideum in yeast (45). The latter enzyme is not known to be subject to any kind of allosteric regulation (46), and yet yeast transformants carrying the gene on multicopy plasmids grow like wild-type cells on media containing either glucose or gluconeogenic carbon sources (45).…”

Section: Figsupporting

confidence: 70%

“…The latter enzyme is not known to be subject to any kind of allosteric regulation (46), and yet yeast transformants carrying the gene on multicopy plasmids grow like wild-type cells on media containing either glucose or gluconeogenic carbon sources (45). Finally, our data underline the importance of allosteric regulation of Pfk for yeast metabolism, a notion established previously in in vitro experiments trying to mimic the in vivo situation as closely as possible (47).…”

Section: Figsupporting

confidence: 57%

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A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

Heinisch

Boles

Timpel

1996

Journal of Biological Chemistry

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In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase. Specifically, we identified two amino acids involved in the binding of the most potent allosteric activator fructose 2,6-bisphosphate. Thus, Ser 724 was replaced by an aspartate and His 859 by a serine in each of the enzyme subunits. Whereas the substitutions had no drastic effects when introduced only in one of the two types of subunits, kinetic parameters were modified when both subunits carried the mutation. Thus, the enzyme with His 859 3 Ser showed an increase in K a for binding of the activator, whereas the one with Ser 724 3 Asp failed to react to the addition of fructose 2,6-bisphosphate, at all. The enzymes still responded to other allosteric activators, such as AMP. Stabilities of the mutant subunits were not significantly altered in vivo, as judged from Western blot analysis. Phenotypically, strains expressing the mutant PFK genes showed a pronounced effect on the level of intermediary metabolites after growth on glucose. Mutants not responding to the activator at all (Ser 724 3 Asp) also displayed higher generation times on glucose medium. This could be suppressed by increasing the gene dosage of the mutant alleles. These results indicate that fructose 2,6-bisphosphate through its activation of phosphofructokinase plays an important role in regulation of the glycolytic flux.

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Section: Figsupporting

confidence: 70%

Section: Figsupporting

confidence: 57%

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

Heinisch

Boles

Timpel

1996

Journal of Biological Chemistry

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“…on May 12, 2018 by guest http://jb.asm.org/ edly the rate of growth of the yeast on glucose (6). It would be wrong, however, to conclude that the corresponding regulatory features are useless.…”

Section: Resultsmentioning

confidence: 99%

The regulatory characteristics of yeast fructose-1,6-bisphosphatase confer only a small selective advantage

Navas

Gancedo

1996

J Bacteriol

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The question of how the loss of regulatory mechanisms for a metabolic enzyme would affect the fitness of the corresponding organism has been addressed. For this, the fructose-1,6-bisphosphatase (FbPase) from Saccharomyces cerevisiae has been taken as a model. Yeast strains in which different controls on FbPase (catabolite repression and inactivation; inhibition by fructose-2,6-bisphosphate and AMP) have been removed have been constructed. These strains express during growth on glucose either the native yeast FbPase, the Escherichia coli FbPase which is insensitive to inhibition by fructose-2,6-bisphosphate, or a mutated E. coli FbPase with low sensitivity to AMP. Expression of the heterologous FbPases increases the fermentation rate of the yeast and its generation time, while it decreases its growth yield. In the strain containing high levels of an unregulated bacterial FbPase, cycling between fructose-6-phosphate and fructose-1,6-bisphosphate reaches 14%. It is shown that the regulatory mechanisms of FbPase provide a slight but definite competitive advantage during growth in mixed cultures.Many metabolic enzymes present regulatory features which should facilitate the coordinate operation of different metabolic pathways in response to changes in the environment. It has therefore been assumed that the acquisition by enzymes of specific regulatory mechanisms confers benefits to the organisms in which they are found. However, a quantitation of the selective advantage provided by a given regulatory feature has been rarely attempted. As the fructose-1,6-bisphosphatase (FbPase) from Saccharomyces cerevisiae is subject to multiple regulatory mechanisms-catabolite repression, catabolite inactivation, and inhibition by AMP and fructose-2,6-bisphosphate (F-2,6-P 2 ) (8, 10, 11)-this enzyme could be used to evaluate the physiological significance of a particular regulatory mechanism. The presence of the gluconeogenic enzyme FbPase in a glucose-growing yeast strain could allow the operation of futile cycles (13) and therefore decrease the fitness of the yeast. However, an engineered yeast strain which can synthesize FbPase during growth on glucose did not differ markedly from a wild-type yeast strain either in its generation time or in its growth yield (13). A possible explanation for this observation was that the FbPase was inhibited by AMP and F-2,6-P 2 and therefore had little activity in vivo. It appeared then worthwhile to remove different controls on FbPase and to examine the characteristics of the corresponding yeast strains.To bypass catabolite repression, the coding region of the corresponding gene can be fused to a promoter not repressed by glucose (13); to overcome other regulatory mechanisms, the structure of the yeast FbPase itself should be modified. Sitedirected mutagenesis cannot be used to obtain a deregulated yeast FbPase, as there is not enough information available about the residues which are important for the sensitivity to catabolite inactivation or for inhibition by F-2,6-P 2 . However, use can be ma...

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“…All mutants were verified by DNA sequencing. For expression in yeast, mutant pfk genes were inserted as BamHI fragments downstream of the PFK2 promoter of Saccharomyces cerevisiae in the plasmid pJJH71 (29).…”

Section: Methodsmentioning

confidence: 99%

Creation of an Allosteric Phosphofructokinase Starting with a Nonallosteric Enzyme

Santamaría

Estévez²,

Martı́nez-Costa

et al. 2002

Journal of Biological Chemistry

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An allosteric phosphofructokinase (PFK) was created by sequence manipulation of the nonallosteric enzyme from the slime mold Dictyostelium discoideum (DdPFK). Most amino acid residues proposed as important for catalytic and allosteric sites are conserved in DdPFK except for a few of them, and their reversion did not modify its kinetic behavior. However, deletions at the unique C-terminal extension of this PFK produced a markedly allosteric enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited hysteresis in the time course, intense cooperativity (n H ‫؍‬ 3.8), and a 200-fold decrease in the apparent affinity for fructose 6-phosphate (S 0.5 ‫؍‬ 4500 M), strong activation by fructose 2,6-bisphosphate (K act ‫؍‬ 0.1 M) and fructose 1,6-bisphosphate (K act ‫؍‬ 40 M), dependence on enzyme concentration, proton inhibition, and subunit association-dissociation in response to fructose 6-phosphate versus the nonhysteretic and hyperbolic wild-type enzyme (n H ‫؍‬ 1.0; K m ‫؍‬ 22 M) that remained as a stable tetramer. Systematic deletions and point mutations at the C-tail region of DdPFK identified the last C-terminal residue, Leu 834 , as critical to produce a nonallosteric enzyme. All allosteric mutants were practically insensitive to MgATP inhibition, suggesting that this effect does not involve the same allosteric transition as that responsible for fructose 6-phosphate cooperativity and fructose bisphosphate activation.

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Functional complementation of yeast phosphofructokinase mutants by the non‐allosteric enzyme from Dictyostelium discoideum

Cited by 22 publications

References 32 publications

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

The regulatory characteristics of yeast fructose-1,6-bisphosphatase confer only a small selective advantage

Creation of an Allosteric Phosphofructokinase Starting with a Nonallosteric Enzyme

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