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Many hormones, neurotransmitters and growth factors are peptides that carry an amide group at their carboxyl terminus which is essential for their biological activity. The amide is formed by hydroxylation of an additional glycine residue present in the biosynthetic precursor and the hydroxyglycine derivative dissociates to form the peptide amide and glyoxylic acid. Recent discoveries have shown that two enzymes are involved that act sequentially.

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The ability of a series of non-peptide carboxylic acids to act as substrates or inhibitors of the peptide-amidating enzyme (peptidyl-glycine hydroxylase) was assessed by determining their ability to reduce the rate of enzymic conversion of D-tyrosyl-valyl-glycine or D-tyrosyl-phenylalanyl-glycine to the corresponding dipeptide amide. The inclusion of a phenyl substituent in a position distal to the carboxyl group promoted the inhibitory action. The inhibition was found to be irreversible when an olefinic double bond, CI or to the carboxyl group, was present in the molecule; the inhibition appeared to be associated with a covalent interaction between the amidating enzyme and the inhibitor. With 4-phenyl-3-butenoic acid the inhibitory properties were manifest only in the presence of cofactors of the enzyme. When 4-phenyl-3-[2-'4C]butenoic acid was used, the radioactivity was shown to be incorporated into protein that co-chromatographed with active enzyme.Incubation of rat thyroid carcinoma CA77 cells in the presence of 4-phenyl-3-butenoic acid led to a decrease in the levels of intracellular amidating activity and of thyrotropin-releasing hormone, an amidated peptide produced by these cells. The inhibitory effects reached a maximum at approximately 15 h after which the enzyme levels returned to the control values even though the concentration of 4-phenyl-3-butenoic acid in the cells remained unchanged. The results indicate that a mechanism exists in these cells for regulation of amidating activity.During the course of a study on peptide amidation [l] it was observed that glyoxylate phenylhydrazone can act as a substrate for the amidating enzyme (peptidyl-glycine hydroxylase), exhibiting a higher apparent affinity for the enzyme than D-tyrosyl-valyl-glycine, a peptide substrate commonly used for assay of the enzyme [2]. Identification of the reaction product, oxalic acid phenylhydrazide, demonstrated that amidation involves an initial hydroxylation reaction at a position a to the terminal carboxyl group. This finding stimulated a search for possible inhibitors of the amidating enzyme in which the peptide bond of its substrates would be replaced by other structures. In this communication we describe the development of an irreversible inhibitor and have examined whether the inhibition it produces can be demonstrated in vivo by studying its effect on the amidating activity in rat thyroid carcinoma CA77 cells. MATERIALS AND METHODS Materials[2-14C]Malonic acid was obtained from N.E.N. Phenylacetaldehyde, diethylamine, 4-phenyl-3-butenoic acid, pentanoic acid, hexanoic acid, hexylsulphonic acid, phenylacetic acid, 3-phenylpropionic acid, 4-phenylbutanoic acid, 5-phenlypentanoic acid, 2-pentenoic acid, 2-butenoic acid, 4-

Recent developments in the study of peptide amidation are reviewed. The main areas covered are assay procedures, purification of amidating enzymes, co-factors and regulation, mechanism and specificity of the amidating reaction, and multiple forms of the amidating enzyme and glycosylation. Discussion is presented on aspects that are poorly understood and new areas open to investigation are indicated.