Comparison of enzymatic semisyntheses of peptide amides: human growth hormone releasing factor and analogs.

Biomedica biochimica acta1991PMID: 1820039

Plain Language Summary

Multiple enzymatic methods for converting growth hormone-releasing factor precursors into their biologically active amide forms were compared, including approaches using a recombinant amidating enzyme, trypsin, and carboxypeptidase Y. The amidating enzyme achieved essentially complete conversion of the glycine-extended precursor, while trypsin-based methods provided moderate yields for both full-length GRF and the superactive sermorelin analog [Ala15]-GRF(1-29)-NH2.

Abstract

Enzymatic semisyntheses of growth hormone releasing factor (GRF), a 44-residue peptide amide hormone, from C-terminal acid precursors, are compared. A recombinant alpha-amidating enzyme was used to convert the glycine-extended precursor, GRF(1-44)-Gly-OH, to GRF(1-44)-NH2 in an essentially quantitative fashion. Trypsin was used to convert the precursors, GRF(1-43)-OH and GRF(1-44)-OH, to GRF(1-44)-NH2 (60 and 15% conversion, respectively) in a 75% v:v N,N'-dimethylacetamide solution containing a large excess of leucine amide. Carboxypeptidase Y catalyzed transpeptidations of the precursors, GRF(1-44)-OH and [Ala44]-GRF(1-44)-OH, to GRF(1-44)-NH2 in aqueous leucine amide solutions were also attempted. The trypsin catalyzed direct amidation of [Ala15]-GRF(1-29)-OH in concentrated ammonium acetate/ammonia buffer (95% 1,4-butanediol cosolvent) to form the superactive analog, [Ala15]-GRF(1-29)-NH2 (ca. 25% conversion at equilibrium), is also described.

Authors

Bongers, J; Offord, R E; Felix, A M; Lambros, T; Liu, W; Ahmad, M; Campbell, R M; Heimer, E P