Mutant studies of yeast phosphofructokinase

Clifton, D; Fraenkel, D G

doi:10.1021/bi00537a037

Cited by 64 publications

(35 citation statements)

References 21 publications

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“…In contrast, the latter showed a temperature-sensitive phenotype not observed for all other mutant combinations. This further substantiates the view that strains harboring only one of the Pfk subunits ferment glucose through the usual glycolytic pathway despite of the lack of in vitro detectable Pfk activity (9,21). The homomeric enzyme formed from only the Pfk1p subunits would be more sensitive to heat inactivation in vivo and the homomeric enzyme formed from only Pfk2p subunits would be less susceptible to allosteric regulation in vivo, i.e.…”

Section: Fig 5 Heat Inactivation Of Pfk From Different Mutant Strainssupporting

confidence: 78%

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

Rodicio¹,

Strauß²,

Heinisch³

2000

Journal of Biological Chemistry

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Yeast phosphofructokinase is a heterooctameric enzyme subject to a complex allosteric regulation. A mutation in the PFK1 gene, encoding the larger ␣-subunits, rendering the enzyme insensitive to allosteric inhibition by ATP was found to be caused by an exchange of proline 728 for a leucine residue. By in vitro mutagenesis, we introduced this mutation in either PFK1 or PFK2 and found that the exchange in either subunit drastically reduced the sensitivity of the holoenzyme to ATP inhibition. This was accompanied by a lack of allosteric activation by AMP, fructose 2,6-bisphosphate, or ammonium and an increased resistance to heat inactivation. Yeast cells carrying either one mutation or both in conjunction did not display a strong phenotype when grown on fermentable carbon sources and did not show any significant changes in intermediary metabolites. Growth on non-fermentable carbon sources was clearly impaired. The strain carrying both mutant alleles was more sensitive to Congo Red than the wildtype strain or the single mutants indicating differences in cell wall composition. In addition, we found single pfk null mutants to be less viable than wild type at different storage temperatures and a pfk2 null mutant to be temperature-sensitive for growth at 37°C. The latter mutant was shown to be respiration-dependent for growth on glucose.

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Section: Fig 5 Heat Inactivation Of Pfk From Different Mutant Strainssupporting

confidence: 78%

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

Rodicio¹,

Strauß²,

Heinisch³

2000

Journal of Biological Chemistry

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“…Yeast Pfk is a heterooctameric enzyme composed of 4␣-and 4␤-subunits, encoded by the genes PFK1 and PFK2 (10,11). The latter have been cloned, sequenced, and deleted from the haploid yeast genome (12,13).…”

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confidence: 99%

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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“…Even in the most pertinent case of pJilc2-4 the soluble PFK I was very much present although it was detectable only at very low concentrations of ATP (table 1). However, a recent report [8] indicated that mutations in either PFKZ or PFK2 gene can lead to a nearly total loss of PFK I activity; of the two alleles of pfk2 reported by these authors, pfl2-I had practically no activity of the soluble phosphofructokinase at 0.2 mM ATP and 5 mM fructose 6-phosphate. The second allele pfilr2-2, in contrast, had no effect on PFK I activity.…”

Section: Discussionmentioning

confidence: 89%

Mutations in the regulatory subunit of soluble phosphofructokinase from yeast

et al. 1984

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Mutant alleles of the gene PFKZ have been obtained that alter the sensitivity to ATP inhibition of the soluble yeast phosphofructokinase. One of the alleles makes the enzyme sensitive to micromolar concentrations of ATP. Intragenic revertants of PFK2 mutants confirm that the PFK2 gene determines not only the regulatory properties of the soluble enzyme but also the catalytic activity of particulate phosphofructokinase.

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Mutant studies of yeast phosphofructokinase

Cited by 64 publications

References 21 publications

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

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

Mutations in the regulatory subunit of soluble phosphofructokinase from yeast

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