Conformational Dynamics and Allostery in Pyruvate Kinase

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Pyruvate kinase (PK) is crucial for glycolysis, with allosteric regulation controlling pathway flux.
• Two models, domain rotation and rigid body reorientation, explain allosteric regulation in E. coli PK type 1.

Purpose of the Study:

• To elucidate the conformational and dynamic changes driving allostery in E. coli PK type 1.
• To differentiate between the domain rotation and rigid body reorientation models of allosteric regulation.

Main Methods:

• Time-resolved electrospray ionization mass spectrometry (tr-ESI-MS) coupled with hydrogen-deuterium exchange (HDX).
• Thermostability studies and mutagenic analysis were employed to validate findings.

Main Results:

• Fructose 1,6-bisphosphate (FBP) binding induces a global increase in enzyme conformational dynamics.
• HDX mapping identified specific regions with altered flexibility, particularly an α-helix bridging allosteric and active sites.
• Increased flexibility at the interdomain interface supports the domain rotation model.

Conclusions:

• FBP binding destabilizes key structural elements, leading to increased flexibility and altered active site conformation.
• The findings support the domain rotation model for E. coli PK type 1 allosteric regulation.
• This study provides a mechanistic explanation for how FBP binding influences the enzyme's active site.