Stănciuc Nicoleta, Aprodu Iuliana, Ioniță Elena, Bahrim Gabriela, Râpeanu Gabriela
"Dunărea de Jos" University of Galati, Faculty of Food Science and Engineering, Domnească Street 111, 800201 Galati, Romania.
"Dunărea de Jos" University of Galati, Faculty of Food Science and Engineering, Domnească Street 111, 800201 Galati, Romania.
Spectrochim Acta A Mol Biomol Spectrosc. 2015 Aug 5;147:43-50. doi: 10.1016/j.saa.2015.03.023. Epub 2015 Mar 20.
Given the importance of peroxidase as an indicator for the preservation of vegetables by heat treatment, the present study is focused on enzyme behavior under different pH and temperature conditions, in terms of process-structure-function relationships. Thus, the process-structure-function relationship of peroxidase was investigated by combining fluorescence spectroscopy, in silico prediction methods and inactivation kinetic studies. The fluorescence spectra indicated that at optimum pH value, the Trp(117) residue is not located in the hydrophobic core of the protein. Significant blue- and red-shifts were obtained at different pH values, whereas the heat-treatment did not cause significant changes in Trp and Tyr environment. The ANS and quenching experiments demonstrated a more flexible conformation at lower pH and respectively at higher temperature. On the other hand molecular dynamics simulations at different temperatures highlighted that the secondary structure appeared better preserved against temperature, whereas the tertiary structure around the heme was more affected. Temperature dependent changes in the hydrogen bonding and ion paring involving amino acids from the heme-binding region (His(170) and Asp(247)) might trigger miss-coordination of the heme iron atom by His(170) residue and further enzyme activity loss.
鉴于过氧化物酶作为蔬菜热处理保鲜指标的重要性，本研究聚焦于不同pH和温度条件下酶的行为，涉及过程-结构-功能关系。因此，通过结合荧光光谱、计算机模拟预测方法和失活动力学研究，对过氧化物酶的过程-结构-功能关系进行了研究。荧光光谱表明，在最佳pH值下，色氨酸（Trp(117)）残基并不位于蛋白质的疏水核心中。在不同pH值下观察到显著的蓝移和红移，而热处理并未导致色氨酸和酪氨酸环境发生显著变化。ANS和猝灭实验表明，在较低pH值和较高温度下，构象更为灵活。另一方面，不同温度下的分子动力学模拟突出显示，二级结构似乎对温度具有更好的耐受性，而血红素周围的三级结构受影响更大。涉及血红素结合区域（组氨酸（His(170)）和天冬氨酸（Asp(247)））氨基酸的氢键和离子配对的温度依赖性变化，可能会引发组氨酸（His(170)）残基导致的血红素铁原子错配，进而导致酶活性丧失。
