RT Journal Article
SR Electronic
T1 Multiple Binding Sites for Substrates and Modulators of Semicarbazide-Sensitive Amine Oxidases: Kinetic Consequences
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 525
OP 538
DO 10.1124/mol.107.040964
VO 73
IS 2
A1 Andrew Holt
A1 David J. Smith
A1 Laura Cendron
A1 Giuseppe Zanotti
A1 Adelio Rigo
A1 Maria Luisa Di Paolo
YR 2008
UL http://molpharm.aspetjournals.org/content/73/2/525.abstract
AB Human semicarbazide-sensitive amine oxidase (SSAO) is a target for novel anti-inflammatory drugs that inhibit enzymatic activity. However, progress in developing such drugs has been hampered by an incomplete understanding of mechanisms involved in substrate turnover. We report here results of a comparative study of human and bovine SSAO enzymes that reveal binding of substrates and other ligands to at least two (human) and up to four (bovine) distinct sites on enzyme monomers. Anaerobic spectroscopy reveals binding of substrates (spermidine and benzylamine) and of an imidazoline site ligand (clonidine) to the reduced active site of bovine SSAO, whereas interactions with oxidized enzyme are evident in kinetic assays and crystallization studies. Radioligand binding experiments with [3H]tetraphenylphosphonium, an inhibitor of bovine SSAO that binds to an anionic cavity outside the active site, reveal competition with spermidine, benzylamine, and clonidine, indicating that these ligands also bind to this second anionic region. Kinetic models of bovine SSAO are consistent with one spermidine molecule straddling the active and secondary sites on both oxidized and reduced enzyme, whereas these sites are occupied by two individual molecules of smaller substrates such as benzylamine. Clonidine and other imidazoline site ligands enhance or inhibit activity as a result of differing affinities for both sites on oxidized and reduced enzyme. In contrast, although analyses of kinetic data obtained with human SSAO are also consistent with ligands binding to oxidized and reduced enzyme, we observed no apparent requirement for substrate or modulator binding to any secondary site to model enzyme behavior. The American Society for Pharmacology and Experimental Therapeutics