马肝酒精脱氢酶 (HLADH) 的动态结构：HLADH-NAD+-PhCH2OH、HLADH-NAD+-PhCH2O- 和 HLADH-NADH-PhCHO 的分子动力学模拟结果,Journal of the American Chemical Society

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马肝酒精脱氢酶 (HLADH) 的动态结构：HLADH-NAD+-PhCH2OH、HLADH-NAD+-PhCH2O- 和 HLADH-NADH-PhCHO 的分子动力学模拟结果
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2001-12-01 , DOI: 10.1021/ja0109747
J Luo ₁ , T C Bruice

Affiliation

已经进行了马肝醇脱氢酶 (HLADH) 氧化苯甲醇的分子动力学模拟。已经研究了以下三种状态：HLADH.PhCH(2)OH.NAD(+) (MD1)、HLADH.PhCH(2)O(-).NAD(+) (MD2) 和 HLADH.PhCHO.NADH ( MD3)。MD1、MD2 和 MD3 模拟是在以活性位点为中心的 TIP3P 水的 32-A 半径球体中覆盖的二聚酶亚基之一上进行的。当 HLADH.PhCH(2)OH.NAD(+) --> HLADH.PhCH(2)O(-).NAD(+) 从活性位点转移到溶剂水中时，醇电离产生的质子通过由丝氨酸 48 羟基、核糖 2'-和 3'-羟基和 Hist51 组成的氢键网络。核糖的 3'OH 与 Ile269 羰基的氢键保持该质子转移到水中的位置。分子动力学模拟已被用于跟踪从 TIP3 水池到活动位点的水 1287，从而展示了水进入活动位点的模式。随着时间的推移，水 1287 在两个不同的位置积累，以接受来自核糖 3'-OH 和来自 His51 的质子。可以确定质子通过的两种结构亚态。在第一个亚基中，His51 的咪唑 Ne2 与烟酰胺核糖 C2'-OH 和氢键距离相邻，用于通过氢键接力系列 PhCH(2)OH...Ser48-OH...Ribose2'-OH 进行质子转移...His51...OH(2)（路径 1）平均为 2.0、2.0 和 2.1 A，并且（对于 His51...OH(2)）最小距离小于或等于 2.5 A。路径 1 的结构是呈现 20% 的时间跨度。而在第二个子状态中，有两种可能的质子通道：路径 1 和路径 2 一样。路径 2 涉及氢键继电器系列 PhCH(2)OH...Ser48-OH...Ribose2'-OH...Ribose3'-OH...His51.OH(2 ) 的平均键合距离分别为 2.0、2.0、2.1 和 2.0 A，并且（对于 His51...OH(2)）最小距离小于或等于 2.5 A（时间跨度的概率为 20%）。在分子动力学模拟过程中，NAD(+) 核糖构象稳定在 C2'-endo-C3'-exo 或 C2'-endo 构象。对于 C2'-endo 构象，第一个和第二个亚态能够持续不同的时间跨度，而对于 C2'-endo-C3'-exo 构象，唯一可能的途径涉及第一个亚态。对于第一个和第二个亚态，His51 咪唑环的核糖-OH 质子和 N epsilon 2 之间距离的波动部分是由 His51 咪唑环的“挡风玻璃刮水器”运动造成的。由于 His-51 的咪唑仅对活性贡献约 10 倍，根据用 Gln 取代后活性的降低估计，质子必须有一条替代途径，无需通过该组氨酸即可进入溶剂。第三种途径涉及核糖 C3'-OH 和 Ile-269。在 MD2 中，用于从 PhCH(2)O(-) 到 NAD(+) 的氢化物转移的近攻击构象异构体 (NAC) 约占 ES 构象异构体的 60%。MD3 在弱碱性 pH 值下的分子动力学研究表明，用于氢化物从 NADH 转移到 PhCHO 的反应性基态构象异构体 (NAC) 占构象异构体的 12 mol%。

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Dynamic Structures of Horse Liver Alcohol Dehydrogenase (HLADH): Results of Molecular Dynamics Simulations of HLADH-NAD+-PhCH2OH, HLADH-NAD+-PhCH2O-, and HLADH-NADH-PhCHO

Molecular dynamics simulations of the oxidation of benzyl alcohol by horse liver alcohol dehydrogenase (HLADH) have been carried out. The following three states have been studied: HLADH.PhCH(2)OH.NAD(+) (MD1), HLADH.PhCH(2)O(-).NAD(+) (MD2), and HLADH.PhCHO.NADH (MD3). MD1, MD2, and MD3 simulations were carried out on one of the subunits of the dimeric enzyme covered in a 32-A-radius sphere of TIP3P water centered on the active site. The proton produced on ionization of the alcohol when HLADH.PhCH(2)OH.NAD(+) --> HLADH.PhCH(2)O(-).NAD(+) is transferred from the active site to solvent water via a hydrogen bonding network consisting of serine48 hydroxyl, ribose 2'- and 3'-hydroxyl groups, and Hist51. Hydrogen bonding of the 3'OH of ribose to Ile269 carbonyl maintains this proton in position to be transferred to water. Molecular dynamic simulations have been employed to track water1287 from the TIP3 water pool to the active site, thus exhibiting the mode of entrance of water to the active site. With time the water1287 accumulates in two different positions in order to accept the proton from the ribose 3'-OH and from His51. There can be identified two structural substates for proton passage. In the first substate the imidazole Ne2 of His51 is adjacent to the nicotinamide ribose C2'-OH and hydrogen bonding distances for proton transfer through the hydrogen bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...His51...OH(2) (path 1) average 2.0, 2.0, and 2.1 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A. The structure for path 1 is present 20% of the time span. And in the second substate, there are two possible proton passages: path 1 as before and path 2. Path 2 involves the hydrogen-bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...Ribose3'-OH...His51.OH(2) with the average bonding distances being 2.0, 2.0, 2.1, and 2.0 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A (20% probability of the time span), respectively. During the molecular dynamics simulation the NAD(+) ribose conformations have stabilized at the C2'-endo-C3'-exo or the C2'-endo conformations. With the C2'-endo conformation the first and second substates are able to persist for different time spans, while with the C2'-endo-C3'-exo conformation the only possible pathway involves the first substate. For both first and second substates the fluctuation of the distances between the ribose-OH protons and N epsilon 2 of His51 imidazole ring is partially contributed by the "windshield wiper" motion of the His51 imidazole ring. Since the imidazole of His-51 contributes only about 10-fold to activity, as estimated from the decrease in activity upon substitution with a Gln, there must be an alternate route for the proton to pass to solvent without going through this histidine. A third pathway involves ribose C3'-OH and Ile-269. In MD2, near attack conformers (NACs) for hydride transfer from PhCH(2)O(-) to NAD(+) represent approximately 60% of E.S conformers. The molecular dynamic study of MD3 at mildly basic pH reveals that reactive ground state conformers (NACs) for hydride transfer from NADH to PhCHO amount to 12 mol % of conformers. In MD3, anisotropic bending of the dihydronicotinamide ring of NADH (average value of alpha(c) = 4.0 degrees and alpha(n) = 0.5 degrees, respectively) is observed.

更新日期：2001-12-01

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