A molecular dynamics exploration of the catalytic mechanism of yeast cytosine deaminase.
A molecular dynamics exploration of the catalytic mechanism of yeast cytosine deaminase.
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DOI：
10.1021/jp044828
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发表时间：
2005-03
期刊：
影响因子：
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通讯作者：
L. Yao;S. Sklenak;Honggao Yan;R. Cukier
中科院分区：
文献类型：
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作者：
L. Yao;S. Sklenak;Honggao Yan;R. Cukier
酵母胞嘧啶脱氨酶（酵母胞嘧啶脱氨酶，酵母胞嘧啶脱氨酶）是一种具有重要生物医学意义的锌金属酶，通过一系列分子动力学模拟研究了酵母胞嘧啶脱氨酶的自由形态及其与反应物（胞嘧啶）、产物（尿嘧啶）、几种反应中间体和中间类似物的络合反应。量子化学计算用于构建具有催化作用的锌离子及其配体（两个半胱氨酸，一个组氨酸和一个水）的模型，通过与晶体结构数据的比较，表明半胱氨酸是去质子化的，组氨酸是单质子化的。模拟结果表明，Glu64在yCD的催化作用中起关键作用。Glu64侧链羧基的旋转可以被质子化或去质子化，这使得它可以作为锌结合水和胞嘧啶以及随后的反应中间体之间的质子穿梭体。自由能方法用于获得这些旋转的势垒，它们足够小，可以在纳秒时间尺度上进行旋转。在反应过程中，胞嘧啶重新定向成一种有利于锌结合氢氧化物的亲核攻击的几何形状。反应产物氨的稳定位置位于活性位点，并评估了与水分子的自由交换能。模拟还揭示了c端和含有Phe114的环的微小运动，这可能对反应物结合和产物释放很重要。
Yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, is investigated by a series of molecular dynamics simulations in its free form and complexed with its reactant (cytosine), product (uracil), several reaction intermediates, and an intermediate analogue. Quantum chemical calculations, used to construct a model for the catalytic Zn ion with its ligands (two cysteines, a histidine, and one water) show, by comparison with crystal structure data, that the cysteines are deprotonated and the histidine is monoprotonated. The simulations suggest that Glu64 plays a critical role in the catalysis by yCD. The rotation of the Glu64 side-chain carboxyl group that can be protonated or deprotonated permits it to act as a proton shuttle between the Zn-bound water and cytosine and subsequent reaction intermediates. Free energy methods are used to obtain the barriers for these rotations, and they are sufficiently small to permit rotation on a nanosecond time scale. In the course of the reaction, cytosine reorients to a geometry to favor nucleophilic attack by a Zn-bound hydroxide. A stable position for a reaction product, ammonia, was located in the active site, and the free energy of exchange with a water molecule was evaluated. The simulations also reveal small motions of the C-terminus and the loop that contains Phe114 that may be important for reactant binding and product release.