EDTA inhibits lactoperoxidase-catalyzed iodide oxidation by acting as an electron-donor and interacting near the iodide binding site.
Ethylenediamine tetraacetate (EDTA) inhibits lactoperoxidase (LPO)-catalyzed rate of iodide oxidation in concentration and pH-dependent manner. A plot of log Kiapp values against various pH yields a sigmoidal curve from which an ionisable group of pKa value 6.0 could be ascertained for controlling the inhibition of catalytically active LPO by EDTA. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation by acting as an electron donor. EDTA al so reduces LPO-compound-11 to the native ferric state by one-electron transfer as evidenced by the spectral shift from 428 to 412 nm. Optical difference spectroscopic studies indicate that EDTA binds to LPO with the apparent equilibrium dissociation constant (KD) of 12 +/- 2 mM at pH 6.5. A plot of log KD values against various pH produces a sigmoidal curve from which an ionisable group of LPO having pKa = 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 KD of LPO-EDTA complex is significantly increased (62 +/- 5 mM) by iodide suggesting that EDTA binds close to the iodide binding site. EDTA also increases the KD 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 competitively as an electron donor by interacting at or near the iodide binding site and these sites are close to the aromatic donor binding site.
Department of Physiology, Indian Institute of Chemical Biology, Calcutta, India.
D K
DK
Bhattacharyya
U
U
Bandyopadhyay
R K
RK
Banerjee
D001665:Binding Sites; D002384:Catalysis; D002614:Chelating Agents / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology*; D004579:Electron Transport; D006735:Horseradish Peroxidase / Q000378:metabolism; D007454:Iodides / Q000378:metabolism*; D007784:Lactoperoxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010084:Oxidation-Reduction
D002614:Chelating Agents; D007454:Iodides; D004492:Edetic Acid; D006735:Horseradish Peroxidase; D007784:Lactoperoxidase