Promoting vibrations in human purine nucleoside phosphorylase.: A molecular dynamics and hybrid quantum mechanical/molecular mechanical study
Promoting vibrations in human purine nucleoside phosphorylase.: A molecular dynamics and hybrid quantum mechanical/molecular mechanical study
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DOI：
10.1021/ja0457563
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发表时间：
2004-12-08
影响因子：
15
通讯作者：
Schwartz, SD
中科院分区：
文献类型：
--
作者：
Núñez, S;Antoniou, D;Schwartz, SD
对人嘌呤核苷磷酸化酶（hPNP）与Immucillin家族中几种过渡态（TS）类似物的晶体学研究表明，O-5 '、O-4'和O-P（亲核磷酸氧）原子的几何排列是不寻常的，它们处于紧密的三氧堆叠中。这些观察结果得到了广泛的实验动力学同位素效应分析的证实。我们提出，蛋白质促进的动力学模式在hPNP导致这个堆栈，集中在核糖基O-4'氧，挤压在一起，推动电子向嘌呤环，稳定oxacarbenium字符的TS。由于N-核苷键在反应过程中断裂，N-7和O-6的pK（a）值因氧堆压缩向嘌呤环排出的电子密度而增加。嘌呤环中增加的电子密度改善了与附近残基的静电相互作用，并促进了质子从溶剂质子或未识别的一般酸中的提取，使嘌呤成为更好的离去基团，并加速催化。经典和混合量子/经典分子动力学（MD）模拟的米氏复合物的hPNP与底物鸟苷和磷酸进行评估的存在下，蛋白质促进振动（PPV）。对水溶液中的基材进行了类似的模拟。在催化部位，O-5 '，O-4'和O-P氧以约1000 Hz的频率振动。125和465 cm（-1），而不存在hPNP时为285 cm（-1）。混合量子力学/分子力学方法被用来评估是否这种酶促振动推动氧在一起耦合到反应坐标，从而对催化有直接的积极影响。从经典的MD模拟中采取的几个快照的磷解反应的势能面表现出显着的差异，在氧压缩。我们的计算表明，与反应坐标偶联的PPV的存在，通过将三个氧中心靠近在一起来影响活性位点的电子改变，并加速hPNP催化的磷酸解反应中的底物周转。
Crystallographic studies of human purine nucleoside phosphorylase (hPNP) with several transition-state (TS) analogues in the immucillin family showed an unusual geometric arrangement of the atoms O-5', O-4', and O-P, the nucleophilic phosphate oxygen, lying in a close three-oxygen stack. These observations were corroborated by extensive experimental kinetic isotope effect analysis. We propose that protein-facilitated dynamic modes in hPNP cause this stack, centered on the ribosyl O-4' oxygen, to squeeze together and push electrons toward the purine ring, stabilizing the oxacarbenium character of the TS. As the N-ribosidic bond is cleaved during the reaction, the pK(a) values of N-7 and O-6 increase by the electron density expelled by the oxygen-stack compression toward the purine ring. Increased electron density in the purine ring improves electrostatic interactions with nearby residues and facilitates the abstraction of a proton from a solvent proton or an unidentified general acid, making the purine a better leaving group, and accelerating catalysis. Classical and mixed quantum/classical molecular dynamics (MD) simulations of the Michaelis complex of hPNP with the substrates guanosine and phosphate were performed to assess the existence of protein-promoting vibrations (PPVs). Analogous simulations were performed for the substrates in aqueous solution. In the catalytic site, the O-5', O-4', and O-P oxygens vibrate at frequencies of ca. 125 and 465 cm(-1), as opposed to 285 cm(-1) in the absence of hPNP. The hybrid quantum mechanical/molecular mechanical method was used to assess whether this enzymatic vibration pushing the oxygens together is coupled to the reaction coordinate, and thus has a direct positive impact on catalysis. The potential energy surface for the phosphorolysis reaction for several snapshots taken from the classical MD simulation showed substantial differences in oxygen compression. Our calculations showed the existence of PPVs coupled to the reaction coordinate, which effect electronic alterations in the active site by pushing the three oxygen centers together in proximity, and accelerate substrate turnover in the phosphorolysis reaction catalyzed by hPNP.