EDTA INHIBITS LACTOPEROXIDASE-CATALYZED IODIDE OXIDATION BY ACTING ASAN ELECTRON-DONOR AND INTERACTING NEAR THE IODIDE BINDING-SITE

Ethylenediamine tetraacetate (EDTA) inhibits lactoperoxidase (LPO)-cat alyzed rate of iodide oxidation in concentration and pH-dependent mann er. A plot of log Ki(app) values against various pH yields a sigmoidal curve from which an ionisable group of pK(a) value 6.0 could be ascer tained for controlling the inhibition of catalytically active LPO by E DTA. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation by acting as an electron donor. EDTA al so reduces LPO-compo und-11 to the native ferric state by one-electron transfer as evidence d by the spectral shift from 428 to 412 nm. Optical difference spectro scopic studies indicate that EDTA binds to LPO with the apparent equil ibrium dissociation constant (K-D) of 12 +/- 2 mM at pH 6.5. A plot of log K-D values against various pH produces a sigmoidal curve from whi ch an ionisable group of LPO having pK(a) = 5.47 could be calculated, deprotonation of which favours EDTA binding. EDTA also binds to LPO-CN - complex indicating its binding site away from heme iron centre. The K-D of LPO-EDTA complex is significantly increased (62 +/- 5 mM) by io dide suggesting that EDTA binds close to the iodide binding site. EDTA also increases the K-D value of LPO-hydroquinone complex from 62 +/- 5 mM to 200 +/- 21 mM indicating that EDTA and aromatic donor binding sites are also close. We suggest that EDTA inhibits iodide oxidation c ompetitively as an electron donor by interacting at or near the iodide binding site and these sites are close to the aromatic donor binding site.