Structure of cross-linked rabbit muscle phosphofructokinase in solution

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Phosphofructokinase (PFK) is a key glycolytic enzyme.
• Understanding PFK's quaternary structure is crucial for its function.
• Previous studies have characterized native PFK states.

Purpose of the Study:

• To investigate the structural and hydrodynamic properties of cross-linked rabbit muscle PFK in tetrameric and octameric states.
• To compare the shapes of cross-linked PFK protomers with native states under various conditions.

Main Methods:

• Hydrodynamic methods, including inelastic light scattering, were used to determine translational diffusion coefficients.
• Small-angle X-ray scattering (SAXS) was employed to analyze the radius of gyration, specific inner surface area, and volume.
• SAXS data were modeled using triaxial geometric bodies to determine enzyme dimensions.

Main Results:

• Cross-linked tetrameric PFK exhibited an asymmetric shape (axial ratio of 2) and dimensions of 131.0 x 131.0 x 65.0 Å.
• Cross-linked octameric PFK was approximately spherical (diameter 120.0 Å), resembling a cube with dimensions 120.0 x 120.0 x 120.0 Å.
• Protomer shape differed between native and cross-linked states, with cross-linked protomers being spherical (radius 33.0 Å) and native protomers in the presence of ATP being elongated (axial ratio 1.8–2.0).

Conclusions:

• Cross-linking induces distinct structural changes in PFK tetrameric and octameric forms.
• The protomer shape of PFK is conformationally flexible and sensitive to ligand binding and cross-linking.
• These findings provide insights into PFK's structural dynamics and allosteric regulation.