Binding of 2-naphthols to D38E mutants of 3-oxo-Δ5-steroid isomerase:: Variation of ligand ionization state with the nature of the electrophilic component
Binding of 2-naphthols to D38E mutants of 3-oxo-Δ5-steroid isomerase:: Variation of ligand ionization state with the nature of the electrophilic component
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DOI：
10.1021/bi9917838
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发表时间：
2000-01-11
期刊：
影响因子：
2.9
通讯作者：
Pollack, RM
中科院分区：
文献类型：
--
作者：
Petrounia, IP;Blotny, G;Pollack, RM
3-氧代-δ（5）-类固醇异构酶（KSI）催化多种3-氧代-δ（5）-类固醇异构化为它们的共轭δ（4）异构体。该机制涉及顺序烯醇化和酮化，其中Asp-38通过二烯醇（酯）中间体将质子从C-4转移到C-6。我们以前曾提出，这种中间体是阴离子的，稳定提供了从Tyr-14和Asp-99的氧的类固醇的直接氢键。在这项工作中，我们分析了取代的2-萘酚，这是类似物的中间体二烯醇，D38 E KSI突变体和相应的双突变体缺乏两个亲电基团（D38 E/Y14 F和D38 E/D99 A）之一的结合。这些萘酚在pH 7下与突变型KSI的结合由修改的Bronsted方程描述：log K-D = alpha（pK（a））+常数，其中K-D是复合物的解离常数。D38 E的高α值（α = 0.87 +/-0.06）表明这些D38 E-萘酚复合物中的负电荷几乎完全位于结合的配体上。相比之下，双突变体的α值（对于D38 E/Y14 F，α = 0.28 +/-0.02;对于D38 E/D99 A，α = 0.25 +/-0.02）与结合萘酚的氧上非常少的负电荷一致。5-硝基-2-萘酚的紫外光谱和与这些突变体结合的马钱素的荧光光谱支持这一解释。将这些结果外推到催化反应中的中间体表明，对于与D38 E的反应，中间体是带负电荷的二烯醇化物，其与Tyr-14和Asp-99两者形成氢键。去除这些氢键供体（Tyr-14或Asp-99）中的任何一个都会导致阴离子的不稳定，并导致二烯醇酶-中间体复合物而不是二烯醇化物。
3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of a variety of 3-oxo-Delta(5)-steroids to their conjugated Delta(4) isomers. The mechanism involves sequential enolization and ketonization, with Asp-38 acting to transfer a proton from C-4 to C-6 through a dienol(ate) intermediate. We have previously proposed that this intermediate is anionic, with stabilization provided from direct hydrogen bonding from Tyr-14 and Asp-99 to the oxygen of the steroid. In this work, we analyze the binding of substituted 2-naphthols, which are analogues of the intermediate dienol, to the D38E KSI mutant and the corresponding double mutants lacking one of the two electrophilic groups (D38E/Y14F and D38E/D99A). The binding of these naphthols to the mutant KSIs at pH 7 is described by the modified Bronsted equation: log K-D = alpha(pK(a)) + constant, where K-D is the dissociation constant of the complex. The high value of alpha for D38E (alpha = 0.87 +/- 0.06) indicates that the negative charge in these D38E-naphthol complexes is localized almost exclusively on the bound ligand. In contrast, values of a fur the double mutants (alpha = 0.28 +/- 0.02 for D38E/Y14F and alpha = 0.25 +/- 0.02 for D38E/D99A) are consistent with very little negative charge on the oxygen of the bound naphthol. Ultraviolet spectra of 5-nitro-2-naphthol and the fluorescence spectra of equilenin bound to these mutants support this interpretation. Extrapolation of these results to the intermediate in the catalytic reaction suggests that for the reaction with D38E, the intermediate is a negatively charged dienolate with hydrogen bonding from both Tyr-14 and Asp-99. Removal of either one of these H-bond donors (Tyr-14 or Asp-99) causes destabilization of the anion and results in a dienol enzyme-intermediate complex rather than a dienolate.