Binding of nucleotides AMP and ATP to yeast phosphofructokinase: Evidence for distinct catalytic and regulatory subunits

Laurent, Michel; Chaffotte, Alain; Tenu, Jean‐Pierre; Roucous, Colette; Seydoux, François

doi:10.1016/0006-291x(78)91617-0

Cited by 36 publications

(8 citation statements)

References 14 publications

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“…Moreover, the square arrangement of 4 identical interacting subunits is consistent with the occurrence of a concerted allosteric transition of yeast phosphofructokinase in the presence of fructose &phosphate, involving only half the number of subunits constituting the enzyme oligomer [4]. Hence, these data, in relation to our binding studies [3,4], speak well in favor of f~ction~ly distinct Q and @ subunits in yeast phosphofructokinase.…”

Section: Discussionsupporting

confidence: 84%

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Octameric structure of yeast phosphofructokinase as determined by crosslinking with disuccinimidyl β‐hydromuconate

et al. 1979

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Section: Discussionsupporting

confidence: 84%

“…Binding experiments [3,4] as well as functional properties of yeast phosphofructokinase [4,5] were indicative of a small number of interacting protomers (3 or 4) equal to only half the number of subunits constituting the enzyme oligomer.…”

Section: Introductionmentioning

confidence: 99%

Octameric structure of yeast phosphofructokinase as determined by crosslinking with disuccinimidyl β‐hydromuconate

et al. 1979

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“…Thus, we should start considering that the asymmetry between the tetramers is an inherent characteristic of the yeast Pfk1 that possesses biological significance. The number of substrate molecules bound to the octameric enzyme under saturating conditions is inconclusive, with one group claiming twice as many as the other (four ATP and four F6P (Laurent et al, 1978 versus eight ATP and eight F6P (Nissler et al, 1977a(Nissler et al, , 1977b). We know from the X-ray model of the double-truncated tetramer that the eukaryotic ATP inhibitor site has evolved from the bacterial ADP effector site, thus creating eight possible effector sites for the ATP molecule.…”

Section: Discussionmentioning

confidence: 97%

The structure of the ATP-bound state of S. cerevisiae phosphofructokinase determined by cryo-electron microscopy

Bárcena

Radermacher

Bär

et al. 2007

Journal of Structural Biology

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Phosphofructokinase (Pfk1, EC 2.7.1.11) plays a key regulatory role in the glycolytic pathway. The combination of X-ray crystallographic and biochemical data has provided an understanding of the different conformational changes that occur between the active and inhibited states of the bacterial enzyme, and of the role of the two bacterial effectors. Eukaryotic phosphofructokinases exhibit a far more sophisticated regulatory mechanism, they are more complex structures regulated by a large number of effectors (around 20). Saccharomyces cerevisiae Pfk1 is an 835 kDa hetero-octamer which shows cooperative binding for fructose-6-phosphate (F6P) and non-cooperative binding for ATP. The 3D structure of the F6P-bound state was obtained by cryo-electron microscopy to 1.1 nm resolution. This electron microscopy structure, in combination with molecular replacement using the bacterial enzyme has helped provide initial phases to solve the X-ray structure of the F6P-bound state 12S yeast truncated-tetramer. Biochemical and small-angle X-ray scattering (SAXS) studies had indicated that Pfk1 underwent a large conformational change upon Mg-ATP binding. We have calculated a reconstruction using reference-based 3D projection alignment methods from 0 degrees images acquired from frozen-hydrated preparations of the enzyme in the presence of Mg-ATP. The ATP-bound structure is more extended or open, and the calculated radius of gyration of 7.33 nm (7.0 nm for F6P) is in good agreement with the SAXS data. There is a substantial decrease in the rotational angle between the top and bottom tetramers. Interestingly, all these changes have arisen from a reorientation of the alpha- and beta-subunits in the dimers. The interface region between the alpha- and beta-subunits is now approximately half the size of the one in the F6P-bound structure. This is the first time that the 3D structure of a eukaryotic Pfk1 has been visualized in its T-state (inhibited-state).

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“…4), allosteric inhibition by ATP seems to be one of the basic regulatory mechanisms. Earlier biochemical approaches determined the number of ATP binding sites per yeast Pfk subunit as either 1 or 2 (38,39). Our previously published work in conjunction with the data presented here strongly argues in favor of two independent binding sites localized on each yeast Pfk subunit.…”

Section: Discussionmentioning

confidence: 99%

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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Binding of nucleotides AMP and ATP to yeast phosphofructokinase: Evidence for distinct catalytic and regulatory subunits

Cited by 36 publications

References 14 publications

Octameric structure of yeast phosphofructokinase as determined by crosslinking with disuccinimidyl β‐hydromuconate

Octameric structure of yeast phosphofructokinase as determined by crosslinking with disuccinimidyl β‐hydromuconate

The structure of the ATP-bound state of S. cerevisiae phosphofructokinase determined by cryo-electron microscopy

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

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