Spectroscopic and binding studies on the interaction of inorganic anions with lactoperoxidase.
The interaction of several inorganic species (SCN-, I-, Br-, Cl-, F-, NO2-, N3-, CN-) with bovine lactoperoxidase was investigated through kinetic and binding studies by using UV-Vis spectroscopy. The above ligands form 1:1 complexes with the protein and can be assigned to three different groups, on the basis of the dissociation constant values (KD) of the adducts: (1) SCN-, I-, Br-, and Cl- (KD increases along the series); (2) F- (which shows a singular behavior); (3) NO2-, N3-, and CN- (that bind at the iron site). KD values for the LPO/SCN- adduct appeared to be modified in the presence of other inorganic species; a strong competition between this substrate and all other anions (with the exception of F-) was evidentiated. Binding investigations on the natural substrates SCN- and I-, at varying pH and temperature, showed that their interaction with lactoperoxidase involves the protonation of a common site in proximity of the iron (possibly distal histidine). Michaelis-Menten constants for SCN-, I-, and Br- followed roughly the same trend as KD; KM for hydrogen peroxide is strongly dependent on the cosubstrate. Computer-assisted docking simulations showed that all ligands can penetrate inside the heme pocket.
Ferrari
R P
RP
Dipartimento di Chimica I.F.M., Università di Torino, Italy. ferrari@ch.unito.it
Ghibaudi
E M
EM
Traversa
S
S
Laurenti
E
E
De Gioia
L
L
Salmona
M
M
D000818:Animals; D000838:Anions / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D001965:Bromides / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D003198:Computer Simulation; D006863:Hydrogen-Ion Concentration; D007454:Iodides / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007784:Lactoperoxidase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D013057:Spectrum Analysis / Q000379:methods*; D013696:Temperature; D013861:Thiocyanates / Q000737:chemistry / Q000378:metabolism
D000838:Anions; D001965:Bromides; D007454:Iodides; D013861:Thiocyanates; D007784:Lactoperoxidase; C031760:thiocyanate