Sequential processing reactions in the formation of hormone amides.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Peptide amidation is crucial for the biological activity of many peptide hormones.
• The enzymatic machinery responsible for prohormone processing, including amidation, is complex and involves multiple steps.

Purpose of the Study:

• To investigate the substrate specificity of a porcine pituitary amidating enzyme.
• To elucidate the sequential order of peptide processing reactions, specifically C-terminal basic residue removal and amidation.

Main Methods:

• Enzyme purification and characterization.
• Substrate specificity assays using synthetic peptides (D-Tyr-Val-Gly and D-Tyr-Val-Gly-Lys-Arg).
• Inhibition studies using carboxypeptidase inhibitors.

Main Results:

• A purified amidating enzyme accepted the tripeptide but not the pentapeptide.
• Both membrane and secretory granule fractions processed the pentapeptide to dipeptide amide after a latency period, suggesting prior cleavage.
• Inhibition studies indicated carboxypeptidase activity precedes amidation of the pentapeptide.

Conclusions:

• Peptide amidation involves a sequential process: C-terminal basic residue removal by carboxypeptidase followed by amidation.
• These two prohormone-processing reactions occur consecutively and do not appear to synergize.