Amidation of growth hormone releasing factor (1-29) by serine carboxypeptidase catalysed transpeptidation.
The applicability of serine carboxypeptidase catalysed transpeptidation reactions, using amino acid amides as nucleophiles, for C-terminal amidation of peptides has been investigated. With the aim of converting an unamidated precursor into GRF(1-29)-NH2, an interesting biologically active derivative of growth hormone releasing factor, a number of model reactions were initially investigated. In such a transpeptidation reaction, where the C-terminal amino acid is replaced by the amino acid amide, used as nucleophile, the C-terminal amino acid residue of the substrate can be chosen freely since it functions as leaving group and does not constitute part of the product. Since the C-terminal sequence of GRF(1-29)-NH2 is -Met-Ser-Arg-NH2 the model reactions Bz-Met-Ser-X-OH (X = Ala, Leu, Arg) + H-Arg-NH2----Bz-Met-Ser-Arg-NH2 + H-X-OH were first studied. With carboxypeptidase Y and X = Ala or Leu the amidated product could be obtained of 98% and 41%, respectively. With carboxypeptidase W-II and X = Arg a yield of no more than 72% could be obtained. The choice of Ala as leaving group in combination with carboxypeptidase Y therefore appeared optimal. With the longer peptide Bz-Leu-Gln-Asp-Ile-Met-Ser-Ala-OH the amidated product could be obtained in a yield of 78%, using carboxypeptidase Y, the only other product being Bz-Leu-Gln-Asp-Ile-Met-Ser-OH, formed due to the competing hydrolysis reaction. The full length peptide GRF(1-28)-Ala-OH was synthesized by the continuous flow polyamide solid-phase method.(ABSTRACT TRUNCATED AT 250 WORDS)
Breddam
K
K
Carlsberg Laboratory, Dept. of Chemistry, Copenhagen, Denmark.
Widmer
F
F
Meldal
M
M
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000378:metabolism*; D013007:Growth Hormone-Releasing Hormone / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D013379:Substrate Specificity
D000577:Amides; D009842:Oligopeptides; D010446:Peptide Fragments; D013007:Growth Hormone-Releasing Hormone; C059148:somatotropin releasing hormone (1-29); D002268:Carboxypeptidases; C022633:serine carboxypeptidase