The present investigation addresses the problem of the binding mode of phenolic inhibitors and the substrate ascorbate to the active site of ascorbate oxidase. The results from both types of compounds indicate that the binding site is located in a pocket near the type 1 copper center. This information is of general interst for blue multicopper oxidases. Docking calculations performed on the ascorbate oxidase–ascorbate complex show that binding of the substrate occurs in a pocket near type 1 Cu, and is stabilized by at least five hydrogen bonding interactions with protein residues, one of which involves the His512 Cu ligand. Similar docking studies show that the isomeric fluorophenols, which act as competitive inhibitors toward ascorbate, bind to the enzyme in a manner similar to ascorbate. The docking calculations are supported by 19F NMR relaxation measurements performed on fluorophenols in the presence of the enzyme, which show that the bound inhibitors undergo enhanced relaxation by the paramagnetic effect of a nearby Cu center. Unambiguous support to the location of the inhibitor close to type 1 Cu was obtained by comparative relaxation measurements of the fluorophenols in the presence of the ascorbate oxidase derivative where a Zn atom selectively replaces the paramagnetic type 2 Cu. The latter experiments show that contribution to relaxation of the bound inhibitors by the type 2 Cu site is negligible.
|Titolo:||Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies|
|Parole Chiave:||Ascorbate oxidase ; Fluorophenols ; Enzyme inhibition ; NMR relaxation ; Docking calculations|
|Settore Scientifico Disciplinare:||Settore CHIM/03 - Chimica Generale e Inorganica|
|Data di pubblicazione:||2004|
|Digital Object Identifier (DOI):||10.1016/j.biocel.2003.10.003|
|Appare nelle tipologie:||01 - Articolo su periodico|