The enzymatic formation of novel bile acid primary amides.
Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O(2)-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The alpha-amidated peptides and the long-chain acylamides are hormones in humans and other mammals. Bile acid glycine conjugates are also substrates for PAM leading to the formation of bile acid amides. The (V(MAX)/K(m))(app) values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (V(MAX)/K(m))(app) value, 3.1 +/- 0.12 x 10(5) M(-1) s(-1) for 3-sulfolithocholylglycine, is 6.7-fold higher than that for d-Tyr-Val-Gly, a representative peptide substrate. The time course for O(2) consumption and glyoxylate production indicates that bile acid glycine conjugate amidation is a two-step reaction. The bile acid glycine conjugate is first converted to an N-bile acyl-alpha-hydroxyglycine intermediate which is ultimately dealkylated to the bile acid amide and glyoxylate. The enzymatically produced bile acid amides and the carbinolamide intermediates were characterized by mass spectrometry and two-dimensional (1)H-(13)C heteronuclear multiple quantum coherence NMR.
King
L
L
Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania 15282, USA.
Barnes
S
S
Glufke
U
U
Henz
M E
ME
Kirk
M
M
Merkler
K A
KA
Vederas
J C
JC
Wilcox
B J
BJ
Merkler
D J
DJ
D001647:Bile Acids and Salts / Q000378:metabolism*; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006038:Glyoxylates / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009097:Multienzyme Complexes; D009842:Oligopeptides / Q000378:metabolism; D010101:Oxygen Consumption; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D006038:Glyoxylates; D009097:Multienzyme Complexes; D009842:Oligopeptides; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D005998:Glycine