Multiple Binding Sites for Substrates and Modulators of Semicarbazide-Sensitive Amine Oxidases: Kinetic Consequences

Abstract

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 [3 H]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.

Isopropylnorsynephrine is a stronger lipolytic agent in human adipocytes than synephrine and other amines present in Citrus aurantium

“…It is also consistent with very ancient findings indicating that the lipolytic response to this adrenergic drug was not differing when using albumins of different origin (human vs. bovine) in the incubation medium, while the potency of catecholamines (e.g., adrenaline) was different from one preparation to another [16]. Our current interpretation of such findings is that, in this period, albumin preparations were less purified than at the present time, and the bovine one was more contaminated by soluble amine oxidase, the activity of which is more abundant in bovine than in human plasma [14]. Consequently, the catecholamines were more oxidized in the former preparation and their lipolytic properties were altered, when compared to incubations with human albumin.…”

Structure and Inhibition of Human Diamine Oxidase

“…There is one structure of a non-covalently bound inhibitor of a mammalian CAO. Clonidine binds to BSAO in a solvent accessible pocket near the active site that forces the TPQ away from its active 'off-copper' conformation and into its 'on-copper' conformation (35). We have shown that clonidine is a weak inhibitor of hDAO.…”

Analgesic efficacy of CR4056, a novel imidazoline-2 receptor ligand, in rat models of inflammatory and neuropathic pain

“…Other purported activities of I2R ligands are related to the modulation of brain creatine kinase (B-CK)8 and semicarbazide-sensitive amino-oxidase (SSAO) 9. The physiological relevance of these molecular targets may suffice to explain the putative involvement of I2R in various diseases10–12 including neuropathic and inflammatory pain 13–16…”