Probing mechanisms for enzymatic activity enhancement of organophosphorus hydrolase in functionalized mesoporous silica.
Probing mechanisms for enzymatic activity enhancement of organophosphorus hydrolase in functionalized mesoporous silica.
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DOI：
10.1016/j.bbrc.2009.10.112
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发表时间：
2009-12-25
影响因子：
3.1
通讯作者：
Liu, Jun
中科院分区：
文献类型：
--
作者：
Chen, Baowei;Lei, Chenghong;Shin, Yongsoon;Liu, Jun
关键词：
我们以前报道过有机磷水解酶（OPH）可以自发地包埋在以HOOC-为官能团的功能化介孔二氧化硅（FMS）中，并且HOOC-FMS中包埋的OPH显示出更高的酶比活性。本工作从OPH-FMS相互作用的角度出发，利用光谱学方法研究了OPH在FMS中表现出增强活性的机制。圆二色谱（CD）结果表明，与游离态OPH相比，HOOC-FMS中OPH的α-螺旋/β-链转变随OPH负载量的增加而增加。色氨酸残基的荧光发射光谱被用来评估酶的三级结构的变化。荧光增加了42%。这与当溶液中尿素浓度降低时，OPH的荧光强度随OPH活性的增加而增加的事实相一致。与溶液中的游离OPH相比，HOOC-FMS中OPH包埋后的稳态各向异性增加，表明包埋后蛋白质的迁移率降低。以丙烯酰胺为碰撞猝灭剂，研究了色氨酸残基的溶剂可及性。与游离OPH相比，OPH-FMS的Trp残基具有较少的溶剂暴露，这是由于其与HOOC-FMS的静电结合，从而显示出增加的荧光强度。这些结果表明，OPH与HOOC-FMS的相互作用导致蛋白质的固定化和OPH在拥挤的限制空间中的有利构象变化，从而增强活性。
We have previously reported that organophosphorus hydrolase (OPH) can be spontaneously entrapped in functionalized mesoporous silica (FMS) with HOOC-as the functional groups and the entrapped OPH in HOOC-FMS showed enhanced enzyme specific activity. This work is to study the mechanisms that why OPH entrapped in FMS displayed the enhanced activity in views of OPH-FMS interactions using spectroscopic methods. The circular dichroism (CD) spectra show that, comparing to the secondary structure of OPH free in solution, OPH in HOOC-FMS displayed increased α-helix/β-strand transition of OPH with increased OPH loading density. The fluorescence emission spectra of Trp residues were used to assess the tertiary structural changes of the enzyme. There was a 42% increase in fluorescence. This is in agreement with the fact that the fluorescence intensity of OPH was increased accompanying with the increased OPH activity when decreasing urea concentrations in solution. The steady-state anisotropy was increased after OPH entrapping in HOOC-FMS comparing to the free OPH in solution, indicating that protein mobility was reduced upon entrapment. The solvent accessibility of Trp residues of OPH was probed by using acrylamide as a collisional quencher. Trp residues of OPH-FMS had less solvent exposure comparing with free OPH in solution due to its electrostatical binding to HOOC-FMS thereby displaying the increased fluorescence intensity. These results suggest the interactions of OPH with HOOC-FMS resulted in the protein immobilization and a favorable conformational change for OPH in the crowded confinement space and accordingly the enhanced activity.
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影响因子：
10.8
作者：
Lei, Chenghong;Shin, Yongsoon;Ackerman, Eric J.
通讯作者：
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