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 (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 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. (C) 2000 Academic Press.