An engineered liver glycogen phosphorylase with AMP allosteric activation

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme kinetics

Background:

• Liver and muscle glycogen phosphorylases are distinct enzymes crucial for glucose homeostasis.
• AMP allosterically activates muscle phosphorylase but not liver phosphorylase efficiently in their unphosphorylated (b) states.

Purpose of the Study:

• To investigate the structural basis for the differential sensitivity of liver and muscle phosphorylase isozymes to AMP.
• To engineer liver phosphorylase for altered AMP responsiveness.

Main Methods:

• Developed a bacterial expression system for liver phosphorylase.
• Created single and double amino acid substitutions in liver and muscle phosphorylase.
• Constructed a chimeric phosphorylase by replacing N-terminal residues of liver phosphorylase with muscle residues.
• Assayed enzyme kinetics, including AMP activation and cooperativity.

Main Results:

• Single amino acid substitutions did not alter AMP responsiveness.
• A chimeric liver phosphorylase (L1M2-48L49-846) showed increased maximal activity upon AMP activation compared to native liver phosphorylase.
• The chimeric enzyme's maximal activity approached that of muscle phosphorylase a.
• AMP binding cooperativity remained unchanged, while heterotropic cooperativity was slightly enhanced.

Conclusions:

• The N-terminal 48 amino acids of phosphorylase modulate intrinsic activity (Vmax), likely through subunit interactions.
• Tissue-specific phosphorylase isozymes utilize complex mechanisms involving N-terminal regions and other undefined regions for allosteric regulation and substrate interaction.
• These findings contribute to understanding the evolution and regulation of glucose homeostasis mechanisms.