Elucidation of amidating reaction mechanism by frog amidating enzyme, peptidylglycine alpha-hydroxylating monooxygenase, expressed in insect cell culture.
A frog 'peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3)' was expressed in cultured insect cells by using the baculovirus expression vector system. The enzyme, recovered in the culture medium, was purified to homogeneity. Its apparent molecular mass (43 kd), estimated by both SDS-PAGE and molecular sieving, was higher than the value (39 kd) for the 'PAM' (AE-I) purified from frog skin. N-terminal sequence analysis indicated that cleavage of signal sequence had occurred but the propeptide still remained at the N terminus. The glycine-extended model peptide X-Gly (mean = Ala-Ile-Gly-Val-Gly-Ala-Pro) was used as substrate for the purified enzyme. The reaction product formed at pH 5.4 was isolated and characterized by amino acid sequence analysis, FAB-MASS and 1H-NMR. It was shown that the purified enzyme had converted the model peptide to the C-terminal alpha-hydroxyglycine-extended peptide [X-Gly(OH)] instead of the amidated product (X-NH2), indicating that the enzyme widely known as 'PAM' should be called 'peptidylglycine alpha-hydroxylating monooxygenase'. A novel enzyme, present in the insect cell culture medium and separable from the expressed monooxygenase, could convert the alpha-hydroxyglycine-extended peptide to the amidated product at physiological pH values. It is concluded that the alpha-amidation of glycine-extended peptides is a two-step process catalyzed by the monooxygenase and the novel enzyme.
Suzuki
K
K
Bio-organics Research Department, CIBA-GEIGY Limited, Takarazuka, Japan.
Shimoi
H
H
Iwasaki
Y
Y
Kawahara
T
T
Matsuura
Y
Y
Nishikawa
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002116:Calcitonin / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics; D009097:Multienzyme Complexes; D012867:Skin / Q000201:enzymology*; D014982:Xenopus laevis / Q000378:metabolism*
D009097:Multienzyme Complexes; D002116:Calcitonin; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D005998:Glycine