Modulating electron density in the bound product, 4-hydroxybenzoyl-CoA, by mutations in 4-chlorobenzoyl-CoA dehalogenase near the 4-hydroxy group.
Modulating electron density in the bound product, 4-hydroxybenzoyl-CoA, by mutations in 4-chlorobenzoyl-CoA dehalogenase near the 4-hydroxy group.
复制标题
通过 4-羟基附近 4-氯苯甲酰-CoA 脱卤酶的突变来调节结合产物 4-羟基苯甲酰-CoA 中的电子密度。
DOI：
10.1021/bi982668k
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Carey,PR
中科院分区：
文献类型：
--
作者：
Dong,J;Xiang,H;Luo,L;Dunaway-Mariano,D;Carey,PR
酶4-氯苯甲酰基-CoA脱卤酶将4-氯苯甲酰基-CoA（4-CBA-CoA）水解为4-羟基苯甲酰基-CoA（4-HBA-CoA）。生物化学和晶体学研究已经确定了脱卤酶残基Asp 145在产物-酶复合物结构中与配体的4-羟基非常接近的关键作用。在本研究中，在Asp 145的位点选择性突变的产物复合物的影响进行了探讨，通过拉曼光谱。WT−产物复合物的光谱特征、λmax的大红移以及拉曼数据中苯甲酰基环模式的完全重组都不存在于D145 E复合物中。WT复合物中的主要光谱扰动是由苯甲酰基羰基处的强电子“拉”和4-OH基团附近的Asp 145侧链的电子“推”引起的。这些因素协同作用，使苯甲酰基的π电子失去活性。由于拉曼数据显示在D145 E络合物中苯甲酰基的羰基处发生非常强的电子牵引，因此很明显，苯甲酰基的4-OH基团附近所需的电子推动缺失。因此，需要将Asp 145的侧链非常精确地定位在苯甲酰基的4-位附近，以引起WT复合物中观察到的显著电子重组，并且具有额外CH 2基团的谷氨酸侧链不能满足该标准。对于另外两种缺乏催化活性的Asp 145突变体D145 A和D145 S，产物复合物的拉曼差光谱数据证明存在一群电离产物（即，4-O-）。离子化酚盐形式的存在解释了这些络合物具有高度红移的吸收最大值（λ max接近400 nm）的观察结果。对于WT复合物，仅观察到4-OH形式，在Asp 145侧链上存在近端负电荷的情况下，电离在能量上是昂贵的。对D145 S和D145 A中结合产物pKa的半定量估计表明，这种电离位于pH 6.5−7.0范围内。这比游离产品的pKa低约2个pH单位。4-二甲氨基苯甲酰基-CoA的拉曼光谱在与脱卤酶结合后发生重大变化。结合形式在1562和1529 cm-1附近具有两个特征，因此非常类似于与野生型酶结合的产物的光谱，这强调了这些复合物中的五重性。新开发的拉曼系统的使用使我们能够在100 - 300 μM范围内获得脱卤酶复合物的正常（非共振）拉曼数据，并预示着拉曼光谱应用于大分子稀溶液的重要进展。
The enzyme 4-chlorobenzoyl-CoA dehalogenase hydrolyzes 4-chlorobenzoyl-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). Biochemical and crystallographic studies have identified a critical role for the dehalogenase residue Asp 145 in close proximity to the ligand's 4-hydroxy group in the structure of the product−enzyme complex. In the present study the effects of site selective mutations at Asp 145 on the product complex are explored by Raman spectroscopy. The spectral signatures of the WT−product complex, the large red shift in λmax, and the complete reorganization of the benzoyl ring modes in Raman data are absent for the D145E complex. The major spectral perturbations in the WT complex are brought about by strong electron “pull” at the benzoyl carbonyl and electron “push” by the side chain of Asp 145 near the 4-OH group. Acting in concert, these factors polarize the benzoyl's π-electrons. Since the Raman data show that very strong electron pull occurs at the benzoyl's carbonyl in the D145E complex, it is apparent that the needed electron push near the benzoyl's 4-OH group is missing. Thus, very precise positioning of Asp 145's side chain near the benzoyl's 4-position is needed to bring about the dramatic electron reorganization seen in the WT complex, and this criterion cannot be met by the glutamate side chain with its additional CH2group. For two other Asp145 mutants D145A and D145S that lack catalytic activity, Raman difference spectroscopic data for product complexes demonstrate the presence of a population of ionized product (i.e., 4-O-) in the active sites. The presence of the ionized phenolate form explains the observation that these complexes have highly red-shifted absorbance maxima with λmaxsnear 400 nm. For the WT complex only the 4-OH form is seen, ionization being energetically expensive with the presence of the proximal negative charge on the Asp 145 side chain. Semiquantitative estimates of the pKafor the bound product in D145S and D145A indicate that this ionization lies in the pH 6.5−7.0 range. This is approximately 2 pH units below the pKafor the free product. The Raman spectrum of 4-dimethylaminobenzoyl-CoA undergoes major changes upon binding to dehalogenase. The bound form has two features near 1562 and 1529 cm-1and therefore closely resembles the spectrum of product bound to wild-type enzyme, which underlines the quinonoid nature in these complexes. The use of a newly developed Raman system allowed us to obtain normal (nonresonance) Raman data for the dehalogenase complexes in the 100−300 μM range and heralds an important advance in the application of Raman spectroscopy to dilute solutions of macromolecules.