The Enzymatic Formation of Novel Bile Acid Primary Amides

• Published in: Archives of biochemistry and biophysics 2000-02, Vol.374 (2), p.107-117
• Main Authors: King, Lawrence, Barnes, Stephen, Glufke, Uta, Henz, Matthias E., Kirk, Marion, Merkler, Kathleen A., Vederas, John C., Wilcox, Benjamin J., Merkler, David J.
• Format: Article
• Language: English
• Subjects: bile acid glycine conjugate; Bile Acids and Salts - metabolism; carbinolamide intermediate; cholylglycine; Glycine - analogs & derivatives; Glycine - metabolism; Glyoxylates - metabolism; Kinetics; Mixed Function Oxygenases - metabolism; Multienzyme Complexes; Oligopeptides - metabolism; Oxygen Consumption; peptidylglycine α-amidating monooxygenase; Substrate Specificity
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1999.1611

Bifunctional peptidylglycine α-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O2-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The α-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 (VMAX/Km)app values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (VMAX/Km)app value, 3.1 ± 0.12 × 105 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 O2 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-α-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 1H–13C heteronuclear multiple quantum coherence NMR.