Chemical modification of phosphorylase b by tetranitromethane. Identification of a functional tyrosyl residue

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Rabbit skeletal muscle glycogen phosphorylase b is a key enzyme in glycogen metabolism.
• Tyrosyl residues are important for enzyme structure and function.
• Tetranitromethane (C(NO2)4) is a chemical reagent used to modify tyrosyl residues.

Purpose of the Study:

• To investigate the role of tyrosyl residues in the activity and allosteric regulation of glycogen phosphorylase b.
• To identify specific tyrosyl residues modified by tetranitromethane and their functional significance.

Main Methods:

• Irreversible inactivation of glycogen phosphorylase b using tetranitromethane.
• Protection experiments with the allosteric activator 5'-AMP.
• Peptide mapping using tryptic digestion, gel filtration, and reverse-phase HPLC.
• Amino acid sequencing to identify modified residues.

Main Results:

• Tetranitromethane irreversibly inactivated glycogen phosphorylase b by nitrating seven tyrosyl residues.
• Inactivation was prevented by 5'-AMP, indicating a role for tyrosyl residues in allosteric activation.
• Tyrosine-75 was identified as the critical residue responsible for activity loss upon nitration.
• Tyrosine-403 and Tyrosine-280 were identified as involved in glycogen storage and glucose binding, respectively, but not essential for catalytic activity.

Conclusions:

• Tyrosine-75 is essential for the catalytic activity of glycogen phosphorylase b.
• Specific tyrosyl residues (Tyr-403, Tyr-280) are accessible and modified but not essential for enzyme function.
• 5'-AMP protects critical tyrosyl residues, highlighting their role in allosteric regulation.