Studies on the regulation of yeast phosphofructo-1-kinase: its role in aerobic and anaerobic glycolysis

Reibstein, David; Hollander, Jan A. den; Pilkis, S J; Shulman, Robert G.

doi:10.1021/bi00349a031

Cited by 68 publications

(67 citation statements)

References 44 publications

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“…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). The genetic approach used here goes one step further in changing only one enzymatic parameter in vivo.…”

Section: Figsupporting

confidence: 59%

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: 59%

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

Heinisch

Boles

Timpel

1996

Journal of Biological Chemistry

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“…Therefore, the amount of radioactive ATP needed to conduct assays at physiological ATP concentrations, which are often in the millimolar range, would be quite high. Some examples of intracellular ATP concentrations include 3 mM for yeast (29), 1 mM in smooth muscle cells (30), and 2.6 mM in human astrocytoma cells (31). We found that the K m values for ATP were in the micromolar range for certain substrates.…”

Section: Discussionmentioning

confidence: 99%

A Carboxyl-terminal Mutation of the Epidermal Growth Factor Receptor Alters Tyrosine Kinase Activity and Substrate Specificity as Measured by a Fluorescence Polarization Assay

Beebe¹,

Wiepz

Guadarrama

et al. 2003

Journal of Biological Chemistry

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The expression of certain COOH-terminal truncation mutants of the epidermal growth factor receptor (EGFR) can lead to cell transformation, and with ligand stimulation, a broader spectrum of phosphorylated proteins appears compared with EGF-treated cells expressing wild-type EGFR. Accordingly, it has been proposed that elements within the COOH terminus may determine substrate specificity of the EGFR tyrosine kinase (Decker, S. J., Alexander, C., and Habib, T. Our data demonstrate that the steady-state kinetic parameters for the ct1022 mutant differ from those of the wild-type enzyme, and the differences are substrate-dependent. These results support the concept that this oncogenic truncation/mutation alters EGFR substrate specificity, rather than causing a general alteration of activity. We performed the experiments using a non-radioactive fluorescence polarization assay that quantifies the degree of phosphorylation of peptide as well as natural substrates. The results are consistent with those from the traditional [␥-32 P]ATP/filtration assay.Binding of EGF 1 and other ligands to EGFR and its relatives (ErbB2, ErbB3, and ErbB4) triggers a highly complicated signaling network (1, 2). Overexpression as well as oncogenic deletions and truncations of EGFR have been observed in many cancer types, including glioblastomas and non-small cell lung, pancreatic, breast, head and neck, colon, prostate, ovarian, and cervical tumors (3-5). The most commonly found mutation (EGFRvIII) involves a deletion in the NH 2 -terminal portion of EGFR, from residues 6 to 273 (5-7). Several other types of rearrangements have been found, however, including even a tandem duplication of the tyrosine kinase and calcium internalization domains (4,5,8). Half of all glioblastomas exhibit EGFR amplification, and of these, 15% possess COOH-terminal truncated EGFR (at residue 958) (5). Furthermore, expressing certain COOH-terminal truncation mutants in cell culture results in dramatic EGF-induced increases in the variety of phosphorylated proteins present compared with those observed in cells expressing wild-type EGFR (9 -12).The cause of this increase in the assortment of phosphorylated proteins has not yet been completely explored; however, EGF-induced receptor internalization and degradation occurred at a slower rate with an oncogenic EGFR mutant possessing a COOH-terminal truncation at residue 973 rather than with wild-type EGFR (10, 13). Cells expressing this mutant develop a transformed phenotype when grown in the presence of low EGF concentrations (13). Moreover, data suggest that ⌬973-EGFR and ErbB2 heterodimers form in cells expressing this mutant. The ErbB2 in these cells is tyrosyl phosphorylated in the absence of added ligand (12). Interestingly, the oncogene product of the avian erythroblastosis virus (verbB) is an NH 2 -and COOH-terminal truncated version of EGFR (ErbB1) (14,15).Cellular changes in phosphotyrosine levels may occur not only as a result of changes in the catalytic properties of the enzyme, but may also reflect alteratio...

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“…Sodium citrate, tested up to 10 mM; sodium acetate, ATP, ADP, AMP, glucose 6-phospahte and fructose 1,6-bisphosphate, tested up to 5 mM; and phosphoenol pyruvate, tested up to 1 raM, did not have any effect on the activities of the purified enzymes of S. cerevisiae and C. utilis. Fructose 2,6-bisphosphate was found to inhibit the enzymes competitively, but only at high concentrations (2-5 mM) which are not to be expected in vivo [20,21]. The affinity of the enzyme of both organisms for pyruvate was determined at three (Table V and Fig.…”

Section: Properties Of Pyruvate Decarboxylasementioning

confidence: 91%

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

Urk

Schipper

Breedveld

et al. 1989

Biochimica et Biophysica Acta (BBA) - General Subjects

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Studies on the regulation of yeast phosphofructo-1-kinase: its role in aerobic and anaerobic glycolysis

Cited by 68 publications

References 44 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

A Carboxyl-terminal Mutation of the Epidermal Growth Factor Receptor Alters Tyrosine Kinase Activity and Substrate Specificity as Measured by a Fluorescence Polarization Assay

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

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