Recombinant type A rat 75-kDa alpha-amidating enzyme catalyzes the conversion of glycine-extended peptides to peptide amides via an alpha-hydroxyglycine intermediate.
The amidation of C-terminal glycine-extended peptides has been analyzed by the use of a truncated type A peptidylglycine alpha-amidating enzyme (alpha-AE) encoded by cDNA prepared with RNA from rat medullary thyroid carcinoma (MTC) cells. Mouse C127 cells transfected with the rat MTC cDNA encoding the truncated type A alpha-AE secrete the expected 75-kDa enzyme into the culture medium. Medium conditioned with the transfected C127 cells converts both dansyl-Tyr-Val-Gly and dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 at levels which are approximately 1000 times higher than the levels found in medium conditioned with untransfected C127 cells. This result indicates that rat type A alpha-AE alone catalyzes a two-step reaction involving an initial hydroxylation of peptidyl-Gly followed by conversion of the peptidyl-alpha-hydroxyglycine intermediate to the amidated product. The involvement of a separate, second enzyme to convert peptidyl-alpha-hydroxyglycine to peptidyl-NH2 is not necessary in this system. The initial hydroxylation step is rate-determining at infinite substrate concentration and requires a reducing equivalent, molecular oxygen, and copper.
Merkler
D J
DJ
Analytical Protein & Organic Chemistry Group, Unigene Laboratories, Inc., Fairfield, New Jersey 07004.
Young
S D
SD
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003470:Culture Media; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes; D010100:Oxygen / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection
D003470:Culture Media; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D010100:Oxygen; D005998:Glycine