Semisynthesis of human growth hormone-releasing factors by alpha-amidating enzyme catalyzed oxidation of glycine-extended precursors.
Recombinant alpha-amidating enzyme was used in the semisynthesis (1-5 mg scale) of human growth hormone-releasing factor, GRF(1-44)-NH2, by in vitro enzymatic oxidation of the glycine-extended precursor, GRF(1-44)-Gly-OH, prepared by solid-phase synthesis. The equipotent analog, GRF(1-29)-NH2, and the superactive analog, [Ala15]-GRF(1-29)-NH2, were also prepared by this route and were fully characterized. Isolated yields of about 75% were obtained, and the products each possessed full potency in an in vitro rat pituitary bioassay and full receptor-binding affinity. Methods to monitor the amidation of polypeptide substrates and analyze the final products are described, including the use of capillary zone electrophoresis. A transient alpha-hydroxyglycine intermediate, [Ala15]-GRF(1-29)-Gly(alpha-OH)-OH, was isolated and characterized. Kinetic studies with this intermediate demonstrate that the rat alpha-amidating enzyme from recombinant mouse C127 cells possesses both the monooxygenase and lyase activities needed to catalyze both steps of the amidation process.
Bongers
J
J
Roche Research Center, Hoffmann-LaRoche Inc., Nutley, NJ 07110.
Felix
A M
AM
Campbell
R M
RM
Lee
Y
Y
Merkler
D J
DJ
Heimer
E P
EP
D000595:Amino Acid Sequence; D002384:Catalysis; D005998:Glycine / Q000032:analysis*; D006038:Glyoxylates / Q000032:analysis; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000096:biosynthesis; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000096:biosynthesis*; D011498:Protein Precursors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017337:Sermorelin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D006038:Glyoxylates; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; C040786:somatotropin releasing hormone (1-44)amide; C077999:somatotropin-releasing hormone (1-29) amide, Ala(15)-; C078015:somatotropin-releasing hormone (1-29), alpha-hydroxy-Gly(14)-Ala(15)-; D017337:Sermorelin; D013007:Growth Hormone-Releasing Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; C031150:glyoxylic acid; D005998:Glycine