Samples Cynthia R, Raushel Frank M, DeRose Victoria J
Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, Texas 77842-3012, USA.
Biochemistry. 2007 Mar 20;46(11):3435-42. doi: 10.1021/bi061951d. Epub 2007 Feb 22.
Phosphotriesterase (PTE) from Pseudomonas diminuta is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphate nerve agents at rates approaching the diffusion-controlled limit. The proposed catalytic mechanism postulates the interaction of the substrate with the metal center and subsequent nucleophilic attack by the bridging hydroxide. X-band EPR spectroscopy was utilized to monitor the active site of Mn/Mn-substituted PTE upon addition of two inhibitors, diisopropyl methyl phosphonate and triethyl phosphate, and the product of hydrolysis, diethyl phosphate. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge were evaluated by measuring changes in the features of the EPR spectra. The EPR spectra support the proposal that the binding of substrate analogues to the binuclear metal center diminishes the population of hydroxide-bridged species. These results, in conjunction with previously published kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the beta-metal weakens the coordination of the hydroxide bridge to the beta-metal. The weakened coordination to the beta-metal ion increases the nucleophilic character of the hydroxide and is coupled to the increase in the electrophilic character of the substrate.
来自微小假单胞菌的磷酸三酯酶（PTE）是一种双核金属酶，它能以接近扩散控制极限的速率催化有机磷酸酯神经毒剂的水解。提出的催化机制假定底物与金属中心相互作用，随后桥连羟基进行亲核攻击。利用X波段电子顺磁共振光谱监测添加两种抑制剂（二异丙基甲基膦酸酯和磷酸三乙酯）以及水解产物磷酸二乙酯后锰/锰取代的PTE的活性位点。通过测量电子顺磁共振光谱特征的变化来评估抑制剂和产物结合对金属中心和羟基桥磁性性质的影响。电子顺磁共振光谱支持底物类似物与双核金属中心结合会减少羟基桥连物种数量的提议。这些结果与先前发表的动力学和晶体学数据相结合，表明通过β-金属处的磷酰氧进行底物结合会削弱羟基桥与β-金属的配位。与β-金属离子配位的减弱增加了羟基的亲核性，并与底物亲电性的增加相关联。
