Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase.
Peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) catalyzes the formation of alpha-amidated peptides from their glycine-extended precursors, thus playing a key role in the processing of peptide neurohormones. We now report that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-dealkylation. Thus, (4-nitrobenzyl)thioacetic acid is converted to the analogous sulfoxide, N-(4-nitrobenzyl)glycine is converted to 4-nitrobenzylamine and glyoxylate, and [(4-nitrobenzyl)oxy]acetic acid is converted to 4-nitrobenzyl alcohol and glyoxylate. All these new activities display the characteristics expected for the normal PAM-catalyzed reductive oxygenation pathway and produce an equimolar amount of glyoxylate together with the heteroatom-containing dealkylation products. The ester [(4-methoxybenzoyl)oxy]acetic acid is not a PAM substrate, but is instead a good competitive inhibitor (KI = 0.48 mM). In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition. Monoethyl fumarate is also a time-dependent inactivator of PAM. Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid. These compounds are the smallest amide substrates yet reported for PAM, and it is thus apparent that only the minimal structure of an acylglycine is required for PAM-catalyzed oxygenative amidation.
Katopodis
A G
AG
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332.
May
S W
SW
D000475:Alkenes / Q000494:pharmacology; D000818:Animals; D001583:Benzoxazoles; D002417:Cattle; D006626:Hippurates; D062385:Hydroxybenzoates / Q000494:pharmacology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009097:Multienzyme Complexes; D013379:Substrate Specificity
D000475:Alkenes; D001583:Benzoxazoles; D006626:Hippurates; D062385:Hydroxybenzoates; D009097:Multienzyme Complexes; C065090:((4-methoxybenzoyl)oxy)acetic acid; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; C030514:hippuric acid