Peptide substrate specificity of the alpha-amidating enzyme isolated from rat medullary thyroid CA-77 cells

The kinetic parameters were obtained for enzymatic alpha-amidation of peptides of the form N-dansyl-(Gly)4-X-Gly-OH, in which the amino acid at position X was substituted with each of the 20 natural amino acids. The enzyme used in these studies was a highly enriched preparation of alpha-amidating enzyme secreted by a clonal (CA-77) cell line which actively expresses mature alpha-amidated peptides. A 130-fold and 11-fold variation respectively in apparent Km and Vmax values was observed. The effect of the amino acid side chain at position X in stabilization of the enzyme-substrate complex decreased through the series X = planar aromatic or sulfur containing greater than neutral aliphatic greater than polar and basic greater than cyclic aliphatic or acidic.