Abstract

Lactoperoxidase, a heme-containing glycoprotein, catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite which acts as an antibacterial agent. The prosthetic heme moiety is attached to the protein through two ester linkages via Glu258 and Asp108. In lactoperoxidase, the substrate-binding site is formed on the distal heme side. To study the effect of physiologically important potassium ion on the structure and function of lactoperoxidase, the fresh protein samples were isolated from yak (Bos grunniens) colostrum and purified to homogeneity. The biochemical studies with potassium fluoride showed a significant reduction in the catalytic activity. Lactoperoxidase was crystallized using 200 mM ammonium nitrate and 20% PEG-3350 at pH 6.0. The crystals of LPO were soaked in the solution of potassium fluoride and used for the X-ray intensity data collection. Structure determination at 2.20 Å resolution revealed the presence of a potassium ion in the distal heme cavity. Structure determination further revealed that the propionic chain attached to pyrrole ring C of the heme moiety, was disordered into two components each having an occupancy of 0.5. One component occupied a position similar to the normally observed position of propionic chain while the second component was found in the distal heme cavity. The potassium ion in the distal heme cavity formed five coordinate bonds with two oxygen atoms of propionic moiety, N^ε2 atom of His109 and two oxygen atoms of water molecules. The presence of potassium ion in the distal heme cavity hampered the catalytic activity of lactoperoxidase.

Graphic abstract

中文翻译：

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钾诱导的乳过氧化物酶部分抑制：乳过氧化物酶与钾离子复合物的结构，分辨率为 2.20 Å

摘要

乳过氧化物酶是一种含血红素的糖蛋白，可催化过氧化氢将硫氰酸盐氧化为用作抗菌剂的次硫氰酸盐。辅血红素部分通过 Glu258 和 Asp108 的两个酯键连接到蛋白质上。在乳过氧化物酶中，底物结合位点形成在远端血红素侧。为研究具有重要生理意义的钾离子对乳过氧化物酶结构和功能的影响，从牦牛（Bos grunniens）中分离新鲜蛋白质样品。) 初乳并纯化至均质。氟化钾的生化研究表明催化活性显着降低。乳过氧化物酶使用 200 mM 硝酸铵和 20% PEG-3350 在 pH 6.0 下结晶。将 LPO 晶体浸泡在氟化钾溶液中，用于 X 射线强度数据收集。2.20 Å 分辨率下的结构测定表明远端血红素腔中存在钾离子。结构测定进一步揭示了连接到血红素部分的吡咯环 C 上的丙酸链被无序地分成两个组分，每个组分的占有率为 0.5。一种成分占据的位置与丙酸链的正常观察位置相似，而第二种成分位于远端血红素腔中。His109的^ε2原子和水分子的两个氧原子。远端血红素腔中钾离子的存在阻碍了乳过氧化物酶的催化活性。

图形摘要