Structural studies of human muscle FBPase.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Muscle fructose-1,6-bisphosphatase (FBPase) is crucial for glucose homeostasis, controlling the gluconeogenic pathway.
• FBPase activity is modulated by divalent cations; calcium ions inhibit the muscle isoform by disrupting the catalytic loop, with residue E69 identified as critical for this sensitivity.

Purpose of the Study:

• To elucidate the structural mechanisms underlying calcium ion inhibition of human muscle FBPase.
• To investigate the role of residue E69 in calcium sensitivity using structural and biochemical approaches.

Main Methods:

• X-ray crystallography of wild-type and E69Q mutant human muscle FBPase in complex with substrate (fructose-1,6-bisphosphate) and product (fructose-6-phosphate).
• Analysis of enzyme quaternary structure and ligand binding within the active site.
• Small-angle X-ray scattering (SAXS) to confirm the enzyme's quaternary structure in solution.

Main Results:

• Five crystal structures revealed conserved binding of substrate and product in the active site for both wild-type and E69Q mutant enzymes.
• The homotetrameric enzyme consistently adopted a cruciform quaternary structure (κ angle of -85°), characteristic of the R-state, irrespective of mutations or ligand presence.
• This R-state quaternary arrangement was validated in solution using SAXS.

Conclusions:

• The study provides detailed structural insights into the inhibition of muscle FBPase by calcium ions, highlighting the role of residue E69.
• The conserved R-state quaternary structure, maintained across different conditions, suggests a stable structural framework for muscle FBPase function and regulation.