Substrates As Probes Of Semicarbazide- Sensitive Amine Oxidase Active Site

The physiological role of semicarbazide-sensitive amine oxidases in the cellular pathways is highly dependent on their interaction with other biomolecules, that are substrates, inhibitors and regulators. The step of molecular recognition is strongly affected both by the structure of the active site and of the biomolecules and by the nature of the interactions (Coulombic and van der Waals forces, hydrogen bonding and hydrophobic effect) between the two “partners”. The knowledge of these factors is fundamental to identify the physiological biomolecules interacting with SSAOs, of particular interest in the case of human SSAO/VAP-1, and to design potential new inhibitors with pharmacological action. Starting from our availability of purified bovine serum amine oxidase (BSAO), the general aim of our studies was to highlight the nature of forces and the chemical- physical factors controlling the docking step of a molecule into the BSAO active site. To probe the characteristics of BSAO active site we used substrates characterized by different chain length and charge distribution (linear monoamines and polyamines). From the dependence of catalytic efficiency (kcat/Km) on pH and ionic strength, it was possible to figure out residues and/or regions of the BSAO active site, which play a key role in the docking of physiological polyamines or monoamines by Coulombic interactions and hydrophobic effect [1-2]. The analysis of the 3D-crystal structure of BSAO [3] and computational docking studies confirmed these findings and permitted to identify them. Furthermore, from the analysis of the kinetic data it was possible to estimate the value the dielectric constant (about 10) for the microenvironment of BSAO active site, value that was confirmed by the using of a fluorescent probe sensitive to the solvent polarity. These information have been the starting point for studies we have recently performed on the human SSAO/VAP-1 since it has 82% of sequence identity with BSAO. The human enzyme is involved in various physio-pathological processes and it is the target of specific inhibitors with pharmacological action. We used solubilised membrane preparations from human adipocytes as source of this enzyme and primary amines, characterized by different size, sterical hindrance and charge distribution as “substrate probes” for the SSAO/VAP-1 active site. Our results suggest that primary amines characterized by an apolar moiety are good substrates, while the presence of a second positive charge at a distance shorter than 10-12 Ǻ from the reactive amino group has an adverse effect the catalytic efficiency, as found in the case of BSAO. Furthermore, from the dependence of the catalytic efficiency on the chain length of aliphatic monoamines, we estimated a dielectric constant of SSAO/VAP-1 active site higher than we have measured for BSAO active site. This characteristic may increase the importance of hydrophobic interactions and decrease that of Coulombic interactions in the docking of a molecule and may help to have a better view of the behaviour of human SSAO/VAP-1 and of bovine serum amine oxidase in relation to their crystallographic structures.