The enzymatic formation of novel bile acid primary amides

Bifunctional peptidylglycine cy-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(delta) 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 H-1-C-13 heteronuclear multiple quantum coherence NMR. (C) 2000 Academic Press.