Bhattacharyya D K, Bandyopadhyay U, Banerjee R K
Department of Physiology, Indian Institute of Chemical Biology, Calcutta, India.
Mol Cell Biochem. 1996 Sep 20;162(2):105-11. doi: 10.1007/BF00227536.
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.
乙二胺四乙酸（EDTA）以浓度和pH依赖性方式抑制乳过氧化物酶（LPO）催化的碘化物氧化速率。将表观抑制常数（Kiapp）的对数值与不同pH值作图，得到一条S形曲线，由此可以确定一个pKa值为6.0的可电离基团，用于控制EDTA对具有催化活性的LPO的抑制作用。动力学研究表明，EDTA作为电子供体竞争性抑制碘化物氧化。EDTA还通过单电子转移将LPO化合物-11还原为天然铁离子状态，这可通过光谱从428nm位移至412nm得到证明。光差光谱研究表明，在pH 6.5时，EDTA与LPO结合，表观平衡解离常数（KD）为12±2 mM。将KD值的对数值与不同pH值作图，得到一条S形曲线，由此可以计算出LPO的一个pKa = 5.47的可电离基团，其去质子化有利于EDTA结合。EDTA还与LPO-CN复合物结合，表明其结合位点远离血红素铁中心。碘化物使LPO-EDTA复合物的KD显著增加（62±5 mM），这表明EDTA靠近碘化物结合位点结合。EDTA还将LPO-对苯二酚复合物的KD值从62±5 mM增加到200±21 mM，表明EDTA和芳香族供体结合位点也很近。我们认为，EDTA作为电子供体通过在碘化物结合位点或其附近相互作用而竞争性抑制碘化物氧化，并且这些位点靠近芳香族供体结合位点。
