Substrate specificity of the gastrin-amidating enzyme

Area of Science:

• Biochemistry
• Molecular Biology
• Endocrinology

Background:

• Gastrin, a peptide hormone, requires a carboxyl-terminal amide for biological activity.
• Progastrin processing involves endoprotease, carboxypeptidase, and amidating enzymes.
• The glycine residue at position 93 (Gly93) is critical for this processing.

Purpose of the Study:

• To investigate the molecular determinants of peptide amidation in vivo.
• To determine the substrate specificity of the amidating enzyme.
• To elucidate the role of the Gly93 residue in progastrin amidation.

Main Methods:

• Mutagenesis of wild-type Gly93 to Ala93 and Ser93 in progastrin.
• Expression of progastrin variants in GH3 and MTC 6-23 endocrine cell lines.
• Incubation of cells in standard medium and in the presence of D-amino acids.

Main Results:

• Mutation of Gly93 to Ala93 or Ser93 abolished amidated gastrin production.
• Incubation with D-alanine or D-serine partially restored amidation for Ala93 and Ser93 mutants, respectively.
• Wild-type progastrin yielded substantial amounts of amidated gastrin.

Conclusions:

• The amidating enzyme exhibits rigid substrate specificity for a glycine-extended precursor.
• The pro-S-alpha-hydrogen of the C-terminal glycine is vital for enzyme-substrate recognition.
• This study highlights the importance of Gly93 in ensuring proper gastrin amidation and biological activity.