Small‐angle X‐ray scattering studies on the quaternary structure of phosphofructokinase from baker's yeast

Plietz, P.; Damaschun, G.; Kopperschl�ger, Gerhard; Müller, Jürgen

doi:10.1016/0014-5793(78)81179-x

Cited by 23 publications

(13 citation statements)

References 6 publications

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“…9 are in complete agreement with the low-resolution model proposed by Plietz et al (1978) (Fig. 1a).…”

Section: Fig 8 (Top)supporting

confidence: 87%

“…However, we were able to obtain more reliable values for these dimensions from our vitreous ice reconstruction: 24 nm in length and 17.0 nm in width. The radius of gyration of 7.0 nm obtained from the last threedimensional reconstruction in ice thresholded using the criterion of maximum steepness of the contour lines is in very good agreement with the values reported from small-angle X-ray scattering experiments (7.4 Ϯ 0.2 nm (Plietz et al, 1978) and 7.0 Ϯ 0.1 nm (Laurent et al, 1984)). …”

Section: Fig 8 (Top)supporting

confidence: 77%

“…The octameric structure of the yeast enzyme was first confirmed by small-angle X-ray scattering experiments (Plietz et al, 1978). Based on their data, the authors were able to propose a model consisting of two tetramers rotated by 63°around their common center of mass, where the subunit arrangement corresponds to a dihedral point group symmetry (Fig.…”

Section: Introductionmentioning

confidence: 96%

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The First Three-Dimensional Structure of Phosphofructokinase from Saccharomyces cerevisiae Determined by Electron Microscopy of Single Particles

Ruíz

Kopperschläger

Radermacher

2001

Journal of Structural Biology

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“…9 are in complete agreement with the low-resolution model proposed by Plietz et al (1978) (Fig. 1a).…”

Section: Fig 8 (Top)supporting

confidence: 87%

Section: Fig 8 (Top)supporting

confidence: 77%

Section: Introductionmentioning

confidence: 96%

See 1 more Smart Citation

The First Three-Dimensional Structure of Phosphofructokinase from Saccharomyces cerevisiae Determined by Electron Microscopy of Single Particles

Ruíz

Kopperschläger

Radermacher

2001

Journal of Structural Biology

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“…The kinetic properties of phosphofructokinase from Saccharomyces cerevisiae, a hetero-octamer composed of two different subunits (a and b) in a b 2 a 4 b 2 configuration, have been extensively characterized (Kopperschläger et al, 1977;Plietz et al, 1978;Tijane et al, 1979). Like its prokaryotic counterpart, the yeast Pfk1 shows a cooperative binding for F6P and a non-cooperative binding for ATP (Reuter et al, 1979).…”

Section: Introductionmentioning

confidence: 98%

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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“…As the yeast Saccharomyces cere isiae is extremely specialized in fermentation (reviewed in [4]), the biochemistry and genetics of its phosphofructokinase have been the subject of numerous investigations (reviewed in [5]). In contrast to the enzyme from most other organisms, which functions as homo-and heterotetramers, the yeast enzyme is a hetero-octamer composed of four α-and four β-subunits, which are arranged as a dimer of tetramers [6][7][8]. The subunits are each subject to limited proteolytic degradation in itro, involving primarily the N-terminal region of about 200 amino acids [9].…”

Section: Introductionmentioning

confidence: 99%

A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase

Kirchberger

Edelmann

Kopperschläger

et al. 1999

Biochemical Journal

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Yeast phosphofructokinase is an oligomeric enzyme whose detectable activity in vitro depends on its hetero-octameric structure. Here we provide data demonstrating that an alanine residue at positions 874 (for the PFK1-encoded α-subunit) or 868 (for the PFK2-encoded β-subunit) is crucial to achieve this structure. Thus subunits carrying substitutions by either aspartate or lysine of this residue cause a lack of phosphofructokinase activity in vitro and signals of the subunits are poorly detectable in Western blots. Size-exclusion HPLC in conjunction with ELISA detection of the enzyme protein confirmed that no functional octamer is produced in such mutants. Our data suggest that the mutant subunits, not being assembled, tend to aggregate and subsequently become degraded. Substitution of the alanine by valine in either subunit leads to a reduction in specific activities, as expected from a conservative exchange. The kinetic data of the latter mutant revealed a higher affinity to the substrate fructose 6-phosphate, a lower extent of ATP inhibition and a lower degree of activation by fructose 2,6-bisphosphate. In addition, the affinity of mutants carrying a valine instead of an alanine in either the α- or the β-subunit to fructose 2,6-bisphosphate was increased. As no X-ray data on eukaryotic phosphofructokinases are available yet, our data provide the first evidence that a non-charge amino acid at position 874 or 868 is essential for the formation of the functional oligomer. This conclusion is substantiated by comparison with the structure of the well-known prokaryotic enzyme.

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Small‐angle X‐ray scattering studies on the quaternary structure of phosphofructokinase from baker's yeast

Cited by 23 publications

References 6 publications

The First Three-Dimensional Structure of Phosphofructokinase from Saccharomyces cerevisiae Determined by Electron Microscopy of Single Particles

The First Three-Dimensional Structure of Phosphofructokinase from Saccharomyces cerevisiae Determined by Electron Microscopy of Single Particles

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

A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase

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