C-terminal region contributes to muscle acylphosphatase three-dimensional structure stabilisation

Area of Science:

• Biochemistry
• Enzymology
• Protein Structure

Background:

• Muscle acylphosphatase is an enzyme involved in cellular signaling.
• The C-terminal region of enzymes often plays critical roles in their structure and function.
• Understanding these roles is essential for protein engineering and drug development.

Purpose of the Study:

• To investigate the catalytic and structural roles of the C-terminal region of muscle acylphosphatase.
• To determine the impact of C-terminal modifications (elongation and deletion) on enzyme activity and stability.

Main Methods:

• Site-directed mutagenesis to create C-terminal elongated (delta+2, delta+4) and deleted (delta-2, delta-3) acylphosphatase mutants.
• Kinetic analysis to assess enzyme activity.
• 1H Nuclear Magnetic Resonance (NMR) spectroscopy to evaluate protein fold and stability.
• Urea inactivation experiments to confirm structural destabilization.

Main Results:

• Deletion of two or three C-terminal residues drastically reduced catalytic activity (to 7% and 4% of wild-type, respectively).
• C-terminal elongation did not significantly alter enzyme behavior.
• All mutants exhibited a native-like fold but showed reduced stability, especially delta-2 and delta-3 mutants.
• Urea inactivation experiments corroborated the reduced stability of C-terminal modified mutants.

Conclusions:

• The C-terminal region of muscle acylphosphatase is vital for stabilizing its three-dimensional structure, particularly around the active site.
• The C-terminal carboxyl group is not directly involved in the catalytic mechanism.
• C-terminal modifications can significantly impact enzyme stability and activity, offering insights for protein engineering.