The amidating enzyme in pituitary will accept a peptide with C-terminal D-alanine as substrate.
A series of tripeptides which terminated in d-alanine, d-serine, d-leucine or l-alanine was synthesized and the peptides tested for their ability to act as substrates for an amidating enzyme present in porcine pituitary. The peptides were allowed to compete with a radiolabelled substrate 125I d-Tyr Phe Gly in the presence of a rate limiting concentration of amidating enzyme and the degree of conversion to 125I d-Tyr Phe amide was determined by ion exchange chromatography. An accelerated procedure was developed for investigating the rates of reaction. The results showed that d-Tyr Phe d-Ala has a significant affinity for the amidating enzyme; no affinity could be demonstrated with d-Tyr Phe 1-Ala, d-Tyr Phe d-Ser or d-Tyr Phe d-Leu. Direct evidence that d-Tyr Phe d-Ala can undergo amidation was obtained by incubating the 125I labelled tripeptide with the pituitary enzyme. Amidation took place readily with d-Tyr Phe d-Ala but not with the other tripeptides; thus, while the enzyme is unable to catalyse the conversion of a peptide terminating in 1-alanine, it can accept a peptide terminating in d-alanine. The results indicate that the amidating enzyme has a highly compact substrate binding site.
D000409:Alanine; D000577:Amides / Q000096:biosynthesis*; D000818:Animals; D008958:Models, Molecular; D009842:Oligopeptides; D010902:Pituitary Gland / Q000201:enzymology*; D011487:Protein Conformation; D013379:Substrate Specificity; D013552:Swine
D000577:Amides; D009842:Oligopeptides; D000409:Alanine