Catechol(amine)s as probes of lactoperoxidase catalytic site structure: Spectroscopic and modeling studies

Binding affinities to lactoperoxidase (LPO) of a homologous series of subst ituted catechol(amine)s [such as catechol, 4-methylcatechol, 3,4-dihydroxyb enzoic acid, 3,4-dihydroxyphenylacetic acid, 3-(3, 4-dihydroxyphenyl)propio nic acid: dopamine, noradrenaline, adrenaline: L-3,4-dihydroxyphenylalanine ] were studied by UV-visible spectroscopy and docking simulations. Dissocia tion constant (K-d) values were calculated by direct fitting of the experim ental data and fall in a range of 3-95 mM. Thermodynamic parameters are com parable with those reported for the interaction of LPO with p-substituted p henols, suggesting a similar general mode of binding. Furthermore, the rela tive contributions to binding energy, described by the unimolecular constan t K-u, show that interaction between protein and ligands originates from a relatively large number of groups. Docking and molecular dynamics simulatio ns, in agreement with experimental evidence, predict that the substrate is localized into the access channel in the vicinity of heme distal pocket. Th is channel is characterized by a hydrophobic patch (six Phe residues) and b y a charged contribution (two Glu and one His residues). All of the substra tes, except caffeic acid, may approach the protein active sire. Positively charged Arg372 acts as a gate above the heme distal pocket and seems to add ress substrate orientation in relation to the side-chain terminal group.