Sequential processing reactions in the formation of hormone amides

Abstract

The substrate specificity of an enzyme with amidating activity, present in porcine pituitary, was investigated by examining its ability to convert the synthetic peptides D-Tyr-Val-Gly and D-Tyr-Val-Gly-Lys-Arg to the dipeptide amide ~-T y r -v a l -C o N H~. The purified enzyme catalysed the amidation reaction with the tripeptide but did not accept the pentapeptide as a substrate. With the mixture of enzymes present in a membrane fraction from porcine pituitary or the enzymes in a secretory granule fraction, both the tripeptide and pentapeptide substrates gave rise to D-Tyr-Val amide; the formation of dipeptide amide from the pentapeptide, however, involved a latency period after which amidation occurred at a similar rate with the two substrates. Evidence was obtained that arginine and lysine were released from the C terminus of the pentapeptide before amidation took place since the rate of formation of dipeptide amide was reduced at pH values that were compatible with amidation but unfavourable to the action of carboxypeptidase H. In addition formation of the dipeptide amide from the pentapeptide was blocked by guanidinoethylmercaptosuccinic acid and glycylarginine, which are inhibitors of carboxypeptidase enzymes.The experiments demonstrate that removal of basic residues from the C terminus of a peptide and amidation at C-terminal glycine are reactions that take place consecutively. These prohormone-processing reactions, which are intrinsic to the formation of hormone amides, did not synergise.The a-amide group of hormone amides is known to be biosynthesised by an enzyme-cdtalysed reaction which involves oxidation of peptides linked to glycine [l, 21. Knowledge of the specificity of the reaction was first gained from studies with synthetic substrates when it was shown that peptides terminating in alanine, leucine, sarcosine, glutamic acid and lysine were not susceptible to amidation whereas peptides terminating in glycine or D-alanine gave rise to the des-glycine or des-D-alanine peptide amide [2, 31. Notably a peptide terminating in the sequence glycyllysine did not undergo amidation, indicating that a glycine COOH group is important for the reaction. These observations, together with the studies of Suchanek and Kreil on the biosynthesis of mellitin [4], have established that a C-terminal glycine residue is essential for enzyme-catalysed amidation and this has been supported by a growing body of evidence that the biosynthetic precursors of hormone amides invariably contain glycine immediately adjacent to the hormone sequence. It has not been established, however, whether the enzyme or enzymes that are involved in amidation might act synergistically with the endopeptidases and exopeptidases that are involved in cleavage and trimming of the prohormone peptide chain prior to the amidation reaction.In a recent study it was reported that a hexapeptide GlpHis-Pro-Gly-Lys-Arg (Glp, pyrrolidonecarboxylyl), which is related to the prohormone of thyrotropin-releasing hormone (TRH) [5], was converted to Glp-His-Pro amid...

Photoperiod‐Dependent Regulation of Carboxypeptidase E Affects the Selective Processing of Neuropeptides in the Seasonal Siberian Hamster (Phodopus sungorus)

“…The antiserum used in this assay is specific for the amidated C‐terminal region of α‐MSH. Amidation of α‐MSH only occurs after enzymatic cleavage by carboxypeptidases ; hence, detected peptide levels directly reflect carboxpeptidase‐dependent enzymatic turnover of ACTH 1–16 to α‐MSH 1–14 .…”

Catalysis of Slow C‐Terminal Processing Reactions by Carboxypeptidase H

“…The enzyme was dissolved before use in 100 mM sodium acetate at pH 5.7 and dilutions of the enzyme were prepared in the same buffer. Secretory granuies were prepared from porcine pituitary by sucrose density centrifugation (Bleakman and Smyth, 1987). The fractions that contained the granules were identified by radioimmunoassay to locate &endorphin (Zakarian and Smyth, 1979) and by enzyme assay to locate amidating activity (Bradbury et al, 1982).…”